Lin28- mediated control of let-7 biogenesis

ABSTRACT

The present embodiments provide for compositions and methods that regulate microRNA-binding protein-mediated miRNA biogensis; for example Lin28-mediated biogenesis of let-7; and in particular Lin28A-recruited 3′ terminal uridylyl transferase (TUTase) uridylation of pre-let-7. A particular embodiment provide compositions and methods for screening for agents that inhibit TUTase-dependent Lin28A-mediated repression of let-7 miRNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a Division of U.S. application Ser. No. 14/269,041,filed May 2, 2014, which is a continuation of U.S. patent applicationSer. No. 14/007,465, filed Sep. 25, 2013, which application is anational stage filing under 35 U.S.C. 371 of International ApplicationPCT/US2012/030497, filed Mar. 26, 2012, which claims the benefit under35 U.S.C. § 119(e) of U.S. Patent Applications No. 61/467,427, filedMar. 25, 2011, and No. 61/562,706, filed Nov. 22, 2011, the contents ofwhich are incorporated herein by reference in their entirety.International Application PCT/US2012/030497 was published under PCTArticle 21(2) in English.

FEDERAL FUNDING

This invention was made with U.S. government support under grant1RO1GM086386, awarded by the National Institute of General MedicalSciences. The U.S. government has certain rights in the invention.

FIELD

The present invention relates to molecular biology. More specifically,the present embodiments provide for compositions and methods thatregulate Lin28-mediated let-7 miRNA biogenesis. A particular embodimentprovides compositions and methods for screening for agents that inhibitTUTase-dependent Lin28A-mediated repression of let-7 miRNA.

BACKGROUND

Cancer claimed the lives of more than 500,000 Americans in 2011.Although the Lin28 oncogenic pathway has been recognized as factor inmany cancerous states, the characterization of the molecular biologyinvolved with Lin28-associated pathogenesis is not well characterized.Moreover, there remains a need for “druggable” targets for therapeuticintervention of the Lin28-mediated oncogenic pathway.

SUMMARY

The present invention provides for compositions and methods to regulatemiRNA biogenesis, in particular for modulating the distinct functions ofLin28A and Lin28B. As shown herein, Lin28B functions in the nucleus andinhibits the miRNA Microprocessor by sequestering primary let-7transcripts. In contrast, Lin28A-mediated inhibition of let-7 expressioninvolves recruitment of a 3′ terminal uridylyl transferase (TUTase)(e.g., Zcchc11/TUTase4/TUT4) to let-7 precursor RNA to block processingby Dicer in the cell cytoplasm. As shown herein, biochemical dissectionand reconstitution assays reveal the TUTase domains necessary andsufficient for Lin28-enhanced pre-let-7 uridylation by, for example,TUTase Zcchc11. In particular, a single C2H2-type zinc finger domain ofZcchc11 is responsible for the functional interaction with Lin28A: Lin28dramatically enhanced C2H2 binding to the terminal loop region ofpre-let-7. Additionally, Zcchc6 (TUTase7) acts as an alternative TUTasethat functions with Lin28A in vitro, and Zcchc11 and Zcchc6 redundantlycontrol let-7 biogenesis in embryonic stem cells. Importantly, theinhibitory effects of Zcchc11 depletion on the tumorigenic capacity andmetastatic potential of human breast cancer cells and xenografts isrestricted to Lin28A-expressing tumors. Overall, the present inventionprovides compositions and methods for modulating the mechanism ofLin28A-mediated TUTase-dependent and Lin28B-mediated TUTase-independentcontrol of let-7 expression in development of stem cells and cancers,supporting the development of new strategies for cancer therapy.

Some embodiments of the present invention relates to compositions andmethods to inhibit TUTases associated with Lin28-enhanced pre-let-7uridylation. Accordingly, some embodiments of the present inventionprovides methods to inhibit Lin28A-enhanced TUTase activity to thusincrease of miRNA biogenesis. Such embodiments are desirable to increasethe level of miRNAs in the cell, such as for example, to increase thelevel of a tumor suppressor miRNAs in a cell, such as the let-7 familyof miRNA molecules. More specific embodiments address the TUTase domainsnecessary and sufficient for Lin28-enhanced pre-let-7 uridylation, forexample by TUTase Zcchc11.

Another embodiment provides compositions and methods for the treatmentor prevention of cancer, by administering to a subject a pharmaceuticalcomposition comprising agents that inhibit the expression and/oractivity of a TUTase that represses miRNA biogenesis, such as a TUTasethat associates with Lin28A to repress let-7 biogenesis (e.g., Zcchc11or Zcchc6). In some embodiments, the subjects have cancer, or are atincreased risk of developing cancer, as indicated by increased levels ofLin28A as compared to a reference level of Lin28A. In furtherembodiments, the subjects are identified to have decreased levels oftumor suppressor miRNA molecules, such as let-7 miRNA, as compared to areference levels of such tumor suppressor miRNA molecule. In someembodiments, a subject amenable to treatment or prevention of cancer isa mammal, for example a human.

In some embodiments, agents useful in the methods and compositions asdisclosed herein for inhibition of TUTase expression (protein or geneexpression) or activity include for example, but are not limited to, asmall molecule, nucleic acid, nucleic acid analogue, aptamer, ribosome,peptide, protein, antibody or a portion, analog, variant, derivative orfragment thereof. In some embodiments, an agent is an antibody, forexample, a recombinant antibody, humanized antibody, chimeric antibody,modified antibody, monoclonal antibody, polyclonal antibody,miniantibody, dimeric miniantibody, minibody, diabody or tribody, orportionsm variants, analogues, fragments or modified versions thereof.In other embodiments, agents useful for inhibition of TUTase expressionare nucleic acid molecules, such as DNA, RNA, nucleic acid analogue,peptide nucleic acid (PNA), pseudo-complementary PNA (pcPNA), lockednucleic acid (LNA), antagomir or analogue thereof. In particularembodiments, an agent can be a RNA molecule, for example a smallinhibitory RNA (RNAi) such as siRNA, microRNA, shRNA, miRNA moleculesand analogues and homologues and variants thereof. In some embodiments,the TUTase is Zcchc11 or Zcchc6.

In some embodiments, a pharmaceutical composition comprising at leastone agent that inhibits TUTase domains necessary and sufficient forLin28A-enhanced pre-let-7 uridylation by, for example, an agent thatinhibits TUTase Zcchc11or Zcchc6, are administered intravenous,intradermal, intramuscular, intraarterial, intralesional, percutaneous,subcutaneous, or by aerosol. In some embodiments, a subject can alsoadministered one or more additional therapies simultaneously, before orafter administration of agents which inhibit the TUTase, for examplesubjects are administered additional therapies such as surgery,chemotherapy, radiotherapy, thermotherapy, immunotherapy, hormonetherapy or laser therapy. For example, a pharmaceutical composition asdisclosed herein comprising at least one agent which inhibits theactivity or expression of TUTase (e.g., Zcchc11or Zcchc6) can beadministered concurrently with, or before, or after, the delivery of alet-7 miRNA, for example where a let-7 miRNA is being used as atherapeutic strategy for cancer in a subject.

Another aspect of the present invention relates to a method for treatingcancer in a subject comprising measuring the level of the expression oractivity of Lin28A in a biological sample obtained from the subject,wherein a clinician reviews the results and if the expression oractivity level of Lin28A is higher than the expression or activity of areference level, the clinician directs the subject to be treated with ananti-cancer therapy and/or a pharmaceutical composition comprising aneffective amount of at least one agent that inhibits the activity and/orexpression of TUTase Zcchc11or Zcchc6.

Another embodiment of the present invention provides for theidentification of small molecule inhibitors of a newly discovered Lin28oncogenic pathway, more specifically the identification of smallmolecules that specifically target the Lin28A pathway to restoreexpression of tumor suppressor let-7 microRNA in cancer. In a particularembodiment, the identification utilizes high throughput screening. Arelated embodiment provides for methods of establishing the efficacy ofthese compounds as potential chemotherapeutics.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show Lin28B regulates let-7 biogenesis through a Zcchc11TUTase-independent mechanism. FIG. 1A shows a schematic representationof human Lin28A and Lin28B. FIG. 1B reflects Western Blot analysis ofZcchc11, Lin28A, and Lin28B in extracts prepared from human cancer celllines. FIG. 1C shows co-immunoprecipitation (co-IP): Hela cells wereco-transfected with Human myc-Lin28A, myc-Lin28B, or myc-Ago2 witheither Flag-Zcchc11 or Flag-EIF6. Flag-IP and Flag- and Myc-westernblots were performed to detect expression and interaction respectively.See also FIG. 2. FIGS. 1D-1G indicate that stable knockdown of Zcchc11leads to upregulation of mature let-7g levels in Lin28A-expressing cellsbut not Lin28B-expressing cell lines. miRNA levels were measured byq.RT-PCR. Error bars represent SEM (n=3). Protein knockdown wasmonitored by Western blot.

FIGS. 2A-2B show that Lin28A physically interacts with Zcchc11. (FIG.2A) Co-immunoprecipitation (co-IP): Hela cells were co-transfected withHuman myc-Lin28A, or myc-Lin28B with a titration of Flag-Zcchc11 plasmid(1×), Flag-Zcchc11 (5×), or control (Mock). Flag-IP and Flag- andMyc-western blots were performed to detect expression and interactionrespectively. (FIG. 2B) Flag-Zcchc11 interacts with endogenous Lin28A.Igrov1 cells were transfected with Flag-Zcchc11 (or Mock) and the IP wasanalyzed by Western blot using antibodies to detect Lin28A.

FIGS. 3A-3F show that Lin28A and Lin28B are differentially localizedwithin the cell. FIG. 3A: Immunoflourescent staining for endogenousLin28A in Igrov1 and Lin28B in H1299 cell lines. Fibrillarin, a knownnucleolar protein, was used as a positive control. FIG. 3B:immunofluorescence analysis of control- and Lin28B-knockdown H1299 celllines.

FIG. 3C: Biochemical fractionation of Igrov1 and H1299 cell lines.Endogenous levels of Lin28A, Lin28B and Zcchc11 in each fraction weredetected by western blot. Fibrillarin was used as a nuclear marker;Tubulin was used as a cytoplasmic marker. FIG. 3D: Schematic of nuclearlocalization signals in the Lin28B protein. FIG. 3E: Localization ofGFP-Lin28 proteins in Hela cells. DGCR8 NLS was used as a positivecontrol for nuclear localization. hrGFP was used as a control for GFPexpression. FIG. 3F: Fractionation of Flag-Lin28 proteins, exogenouslyexpressed in Hela cells. Proteins were detected by Flag western blot.The same control proteins were used as in the fractionation in FIG. 3C.

FIGS. 4A-4C show that Lin28B directly associates with pri-let-7microRNAs. FIG. 4A: pri-let-7 microRNAs accumulate and mature let-7levels decrease in Hela cells overexpressing Lin28B. pri-microRNA andmature microRNA levels were analyzed by q.RT-PCR. FIG. 4B: Levels ofpri-let-7g are higher in Lin28B-expressing cancer cell lines incomparison to Lin28A-expressing cell lines. Levels of pri-let-7g weredetected by qPCR. FIG. 4C: RNA-Immunoprecipiation (RIP) of RNAassociated with exogenously expressed immunopurified Flag-Lin28A, Lin28Band Lin28BΔNLS#1 from Hela cells. RNA was extracted from IP material andanalyzed by q.RT-PCR. Ability of all three immunopurified proteins tobind to pre-let-7 microRNAs equally was validated by EMSA.

FIGS. 5A-5C demonstrate that Lin28B localizes to nucleoli whereMicroprocessor is absent. FIG. 5A: Co-localization of Microprocessorcomponents GFP-Drosha and mCherry-DGCR8 in Hela cells reveals theirdistribution throughout the nucleus and exclusion from nucleoli. FIG.5B: Localization of GFP-Lin28A, Lin28B, or mutant Lin28B proteins withmCherry-DGCR8 in Hela cells reveals non-overlapping localization ofLin28B and DGCR8. FIG. 5C: Fractionation of a Flag-Lin28B Hela stablecell line. Flag-Lin28B and endogenous DGCR8 were detected western blotand shows a non-overlapping subcellular localization of Lin28B and theMicroprocessor. Fibrillarin was used as a control for nucleolarlocalization.

FIGS. 6A-6E show that Lin28B blocks processing of pri-let-7 microRNAs.(FIG. 6A) Schematic of the dual luciferase vector with pri-microRNAsequence 3′ of the Renilla luciferase ORF. FIG. 6B: Transient knockdownof DGCR8 in Hela cells with siRNA. Effect of knockdown was detected bywestern blot. FIG. 6C: Knockdown of DGCR8 leads to stabilization ofdifferent pri-microRNAs, detected as an increased Renilla luciferasesignal. Firefly luciferase signal was used as a normalizer. FIG. 6D:Overexpression of Lin28B in Hela cells leads to stabilization ofpri-let-7 microRNAs, but not pri-miR-125. Levels of pri-miRNAstabilization were detected as increased Renilla luciferase signal as inFIG. 6C. FIG. 6E: Pri-let-7 microRNAs are stabilized more efficiently inLin28B-expressing cell line, H1299, than Lin28A-expressing cell line,Igrov1. Renilla/Firefly ratios for each pri-microRNAs were normalized tothe stabilization of each pri-microRNA in Hela cells, which lack anyLin28 protein.

FIGS. 7A-7F present data showing that Lin28B directly binds andsequesters pri-let-7. FIG. 7A: Colloidal Blue staining of purifiedrecombinant His-Lin28A and His-Lin28B proteins. (FIG. 7B) Binding ofr.Lin28A and r.Lin28B to pre-let-7g was assessed by EMSA performed with0.5 nM 5′-end labeled pre-let-7g RNA and the indicated concentration ofrecombinant protein. Band intensities were quantitated from threeindependent experiments and represented as the fraction of boundpre-let-7g RNA in the plots. Values are given as average ±S.E.M. (n=3).See also FIG. 8. FIG. 7C: EMSA performed indicated concentration ofr.Lin28A and r.Lin28B with in vitro transcribed uniformly labeledpri-let-7g. FIG. 7D: RNA-Immunoprecipitation (RIP) analysis of RNAassociated with immunopurified Flag-Lin28A and Flag-Lin28B from Helacells. RNA was extracted from IP material and analyzed by q.RT-PCR.Error bars±S.E.M. (n=3). Lower panel indicates relative Lin28A andLin28B expression levels by Flag-Western blot. FIG. 7E: Accumulation ofpri-let-7 by transient Lin28B expression in Hela cell detected byq.RT-PCR. Error bars±S.E.M. (n=3). Lower panel indicates relativeexpression levels of Lin28A and Lin28B proteins detected by Flag-Westernblot in transfected cells. FIG. 7F: pri-let-7 accumulates (top panel)and mature let-7 levels decrease (bottom panel) in Hela cells stablyoverexpressing Lin28B. Error bars±S.E.M. (n=3)

FIG. 8 demonstrates stoichiometry shifts of Lin28A and Lin28B. The dataare plotted as the fraction of bound labeled RNA versus molarequivalents of recombinant protein. The 7:1 curve most closelyapproximates the data for both proteins.

FIGS. 9A-9J show that Lin28B-mediated transformation and tumorigenicityis Zcchc11-independent. FIG. 9A: Lin28B mRNA expression levels areincreased during transformation of MCF10A ER-Src cells. Lin28B mRNAexpression was evaluated at 1, 4, 12, 24, 36 hours post-tamoxifen (TAM)treatment of MCF10A ER-Src cells by real-time PCR analysis. Errorbars±S.E.M. (n=3) FIG. 9B: Inhibition of Lin28B expression does notblock the transformation ability of MCF10A ER-Src cells. Phase-contractimages of MCF10A ER-Src treated with TAM for 36 h in the presence orabsence of a siRNA against Lin28B (siLin28B), a monoclonal antibodyagainst IL6 (Ab-IL6) or a siRNA against Zcchc11 (siZcchc11#1). FIG. 9C:Inhibition of Lin28B but not Zcchc11 blocks the tumorigenicity of MCF10AER-Src cells. MCF10A ER-Src transformed cells untreated or treated withsiRNA negative control (siRNA NC), two different siRNAs against Zcchc11(siZcchc11#1, siZcchc11#2), Ab-IL6 and siLin28B were plated in soft agarand their ability to form colonies was evaluated 20 days later. Theexperiment was repeated thrice and the statistical significance wascalculated using Student's t test. FIG. 9D: Effectiveness of siRNAinhibition of Zcchc11 expression in MCF10A ER-Src cells. MCF10A ER-Srccells were treated with siRNA NC or siZcchc11#1 or siZcchc11#2 andZcchc11 mRNA expression was tested by real-time PCR 24 hourspost-transfection. Error bars±S.E.M. (n=3) (FIG. 9E) Inhibition ofLin28B but not of Zcchc11 allows up-regulation of let-7a microRNAexpression in MCF10A ER-Src transformed cells. Let-7a expression levelwas tested by real-time PCR analysis 24 h post transfection. Errorbars±S.E.M. (n=3) (FIG. 9F) Inhibition of Lin28B but not of Zcchc11results in increased IL6 production levels expression in MCF10A ER-Srctransformed cells. IL6 production was examined by ELISA assay in MCF10AER-Src transformed cells 48 h post transfection. mean±SD n=3 (FIG. 9G)Inhibition of Lin28B but not of Zcchc11 suppresses MCF10A ER-Src tumorgrowth in xenografts. The treatments with siRNA NC, siZcchc11#1 andsiLin28B were performed intraperitoneally (i.p.) for 5 cycles startingon day 15. Error bars±S.E.M. (n=3). (FIGS. 9H and 9I) Efficiency ofsiRNA inhibition of Zcchc11 in xenograft tumors (day 39). Zcchc11 mRNAexpression levels were tested by real-time PCR on tumors untreated ortreated with siRNA NC or siZcchc11#1. Error bars±S.E.M. (n=3) (FIG. 9J)Inhibition of Lin28B but not of Zcchc11 allows up-regulation of let-7aexpression levels in xenograft tumors (day 30). Let-7a expression levelswere tested by real-time PCR on tumors untreated or treated with siRNANC or siZcchc11#1 or siLin28B. Error bars±S.E.M. (n=3)

FIGS. 10A-10G demonstrate that Zcchc11 inhibition blocks tumorigenicityand invasiveness of Lin28A-expressing breast cancer cells. FIG. 10A:q.RT-PCR analysis of Zcchc11 knockdown in MDA-MB-231 and T47D breastcancer cells. Error bars±S.E.M. (n=3) FIG. 10B: Inhibition of Zcchc11expression does not affect let-7a expression in Lin28B-expressing cells(MDA-MB-231), while it up-regulates let-7a expression inLin28A-expressing cells (T47D). Let-7a expression levels measured byq.RT-PCR in cells treated with siRNAs for 48 hours. Error bars±S.E.M.(n=3) FIG. 10C: Inhibition of Zcchc11 expression did not affect thecolony formation ability of MDA-MB-231 cells, but suppressed the colonyformation ability of T47D cells. The number of colonies was evaluated 20days post plating in soft agar. The experiment was repeated thrice andthe statistical significance was calculated using Student's t test. FIG.10D: Inhibition of Zcchc11 expression did not affect the invasiveness ofMDA-MB-231 cells, but suppressed the invasive ability of T47D cells. Thenumber of invasive cells was measured 16 hr post transfection withindicated siRNAs. In all assays, ten fields per insert were scored andSD was measured. The experiment was repeated thrice and the statisticalsignificance was calculated using Student's t test. FIG. 10E: Inhibitionof Zcchc11 expression did not suppress tumor growth of MDA-MB-231 cellsin xenografts, however it inhibited tumor growth of T47D cells inxenografts. The treatments with indicated siRNA were performedintraperitoneally (i.p.) for five cycles starting on day 15. Eachtreatment group consisted of five mice. FIG. 10F: q.RT-PCR analysis ofsiRNA inhibition of Lin28B, Lin28A, and Zcchc11 in xenograft tumors (day30) derived from MDA-MB-231 and T47D cells. Error bars±S.E.M. (n=3).FIG. 10G: Inhibition of Lin28B but not of Zcchc11 allows up-regulationof let-7a expression levels in MDA-MB-231 xenograft tumors (day 30).Inhibition of Lin28A or Zcchc11 results in let-7a up-regulation in T47Dxenograft tumors. Let-7a expression levels measured by q.RT-PCR ontumors untreated or treated with indicated siRNA. Error bars±S.E.M.(n=3).

FIGS. 11A-11E demonstrate that inhibition of Zcchc11 expressionsuppresses tumor growth of Lin28A- but not Lin28B-expressing xenografts.(FIGS. 11A-11B) Xenograft experiments were performed with a variety ofdifferent human cancer cell lines. Mice were treated with the indicatedsiRNA for 5 cycles starting on day 15. For all cells lines tested eachtreatment group consisted of 5 mice. While inhibition of Lin28A orLin28B suppressed tumor growth in the relevant xenografts, inhibition ofZcchc11 inhibited growth only of Lin28A- but not Lin28B-expressingtumors. Error bars±S.E.M. (n=3) (FIG. 11C) Analysis of siRNA inhibitionof Zcchc11 in xenograft tumors (day 30) derived from the indicatedcells. (FIG. 11D) Analysis of siRNA inhibition of Lin28B in xenografttumors (day 30) derived from the indicated cells. (FIG. 11E) Analysis ofsiRNA inhibition of Lin28A in xenograft tumors (day 30) derived fromIGROV1 cells. mRNA expression levels were measured by q.RT-PCR on tumorsuntreated or treated with the indicated siRNA. Error bars±S.E.M. (n=3)

FIGS. 12A-12F depict Lin28A and Lin28B expression in primary humancancers. (FIG. 12A) q.RT-PCR analysis of Lin28A, Lin28B and let-7aexpression levels in normal and colon cancer tissues. Tumor samples werefurther classified into two groups expressing either high Lin28A orLin28B. Data expressed as mean±SE. n=3. (FIG. 12B) Immunohistochemistryfor Lin28A, Lin28B and in situ hybridization for let-7a and U6 in normalcolon tissues and colon adenocarcinomas. (FIG. 12C) q.RT-PCR analysis ofLin28A, Lin28B and let-7a in human normal and breast cancer tissues.Tumor samples were further classified into two groups expressing eitherhigh Lin28A or Lin28B. Data expressed as mean±SE. n=3. (FIG. 12D)Lin28A, Lin28B and let-7a expression levels in different breast cancersubtypes. (FIG. 12E) Correlation between Lin28A and Lin28B mRNA levelsassessed by q.RT-PCR with NF-κB phosphorylation status assessed by ELISAassay. (FIG. 12F) Heatmap representation of Lin28A and Lin28B incarcinomas of different origin measured by q.RT-PCR.

FIGS. 13A-13C show that Lin28A, Lin28B and let-7a expression in colonadenocarcinomas. FIG. 13A: A subset of colon adenocarcinomas expressessignificantly higher levels of Lin28A compared to the uninvolved normaltissues. Sections were subjected to immunohistochemistry for Lin28A andcounterstained with haematoxylin. FIG. 13B: A different subset ofadenocarcinomas expresses significantly higher levels of Lin28B comparedto the uninvolved tissues. Sections were subjected toimmunohistochemistry for Lin28B and counterstained with haematoxylin.FIG. 13C: Let-7a levels are decreased in colon adenocarcinomas incomparison to normal tissue. Sections were subjected to in situhybridization for let-7a and counterstained with nuclear fast red. Bar,50 μm.

FIGS. 14A-14C present the domains of Zcchc11 required for Lin28-mediatedpre-let-7 uridylation. FIG. 14A: Schematic representation of Zcchc11 andtruncations used for in vitro uridylation assays. FIG. 14B: Uridylationassays with synthetic pre-let-7g carried out using Flag immunopurified(IP) Zcchc11 variants and IP Lin28. α-Flag Western blots show similaramounts of IP Zcchc11 within experiments. FIG. 14C: Summary of Zcchc11domain requirements from in vitro uridylation assays.

FIG. 15 indicates that Lin28 and Zcchc11 are sufficient for pre-let-7uridylation. Zcchc11 truncation ΔPneumoG/C purified from either HEK293T(IP) or E. coli (Recombinant) was incubated with either Flag-Lin28 (IP)or 6×-His Lin28 (r.Lin28) in a uridylation assay with syntheticpre-let-7g. Below is a schematic representation of the domains presentin ΔPneumoG/C.

FIGS. 16A-16C demonstrate that Lin28 and the C2H2 domain of Zcchc11synergistically bind pre-let-7. FIG. 16A: Colloidal blue stain ofrecombinant Lin28A and recombinant Zcchc11 C2H2 zinc finger domain.(FIG. 16B) EMSA experiment using the indicated recombinant protein and0.5 nM 5′end-labeled pre-let-7g. FIG. 16C: EMSA using subsaturatingbinding conditions with the indicated recombinant proteins and 5 nM 5′end-labeled preE-let-7g.

FIGS. 17A-17B show evidence that the preE of let-7 is sufficient todirect both Lin28 binding and uridylation of pre-let-7. FIG. 17A:Diagram of synthetic RNAs used for in vitro uridylation assays.Pre-let-7g: endogenous precursorlet-7g miRNA sequence. Pre-miR-21:endogenous precursor miR-21 sequence. Pre-21S7L: synthetic RNAconsisting of the let-7g preE and miR-21 stem sequences. FIG. 17B: Left:5′end-labeled RNAs showing equal amounts. Right: Uridylation assay usingWT Flag IP-mZcchc11, with or without Flag IP-mLin28, and the indicatedprecursor miRNAs.

FIGS. 18A-18C show that Zcchc11 and Zcchc6 have a highly similar domainorganization. FIG. 18A: Schematic showing the domain similaritiesbetween hZcchc11 and hZcchc6 with the N-terminal C2H2 zinc fingerhighlighted and critical zinc finger residues in bold. FIG. 18B:Uridylation assay with Flag-IP WT mZcchc11 and a mutant harboring pointmutations in two conserved asparates required for catalysis, with orwithout Flag-IP Lin28. FIG. 18C: Alignment of the nucleotidyltransferase (Ntr) domains of hZcchc11 and hZcchc6. Aspartic acidresidues critical for catalysis are boxed.

FIGS. 19A-19D demonstrate that Zcchc11 and Zcchc6 can both mediateLin28-dependent pre-let-7 uridylation in vitro. FIG. 19A: α-Flag WBshowing relative amounts of Flag-hZcchc11 and Flag-hZcchc6 (left) orFlag-hLin28A and Flag-hLin28B (right). FIG. 19B: EMSA showing similaramounts of functional Flag-hLin28A and Flag-hLin28B used in uridylationassays. (FIG. 19C) Uridylation assays using Flag-hZcchc11 orFlag-hZcchc6 with either Flag-hLin28A or Flag-hLin28B. (FIG. 19D)Uridylation assay with Flag-hZcchc6 and r.Lin28A.

FIGS. 20A-20B show that Zcchc11 and Zcchc6 function redundantly tosuppress let-7 expression in embryonic cells. FIG. 20A: q.RT-PCRanalysis of mature let-7g and mature miR-21 levels in P19 embryonalcarcinoma cells after transfection with the indicated siRNAs (left).mRNA levels of the indicated genes in P19 cells after transfection withthe indicated siRNAs (right). FIG. 20B: qRT-PCR as in FIG. 20A in V6.5mouse embryonic stem cells. For all experiments, miRNA levels werenormalized to sno-142 and mRNA levels were normalized to β-actin. Errorbars represent s.d. of experiments in triplicate.

FIGS. 21A-21B present data from RNA sequence requirements forZcchc11-catalyzed uridylation of mature miRNAs (FIG. 21A) 5′-end labeledRNAs showing equal amounts of RNA. Uridylation assays using Flag-Zcchc11and the indicated RNA. (FIG. 21B) 5′-end labeled RNAs showing equalamounts of RNA and Uridylation assays using Flag-Zcchc11 and theindicated RNA.

FIG. 22 is a bar graph showing evidence for extensive 3′ uridylation ofmature miRNAs. A published data set was analyzed for evidence of 3′terminal nucleotide additions for selected miRNA. The bar graph showsthe percentage of each miRNA with a terminal (1 or more) nucleotideaddition. Sequence reads represent grouped data obtained from all themouse developmental stages and tissues analyzed in that study.

FIGS. 23A-23C present a let-7 sensitive reporter gene. FIG. 23A is aschematic representation of reporter constructs. FIG. 23B showsluciferase activity assay performed on cells transfected with either acontrol vector or one with the let-7 reporter. Below is a Northern Blotof let-7 levels. FIG. 23C shows luciferase activity assay performed onStable H1299 cell line was transfected with 1 μg control plasmid or 1 μgpri-let-7g stem/miR-21 loop plasmid. Luciferase activity was measured 2days after transfection. N=3.

FIGS. 24A-24B demonstrate that depletion of Lin28-Zcchc11 leads toincreased let-7 expression in human cancer cell lines. FIG. 24A showsWestern blot analysis of Lin28A, Lin28B and Zcchc11 in various humancancer cell lines. FIG. 24B is q.RT-PCR analysis of let-7g and miR-21levels after Lin28A, or Zcchc11 knockdown in IGROV1 ovarian cancercells.

FIG. 25 is a scheme for a high throughput screen assay to monitor TUTaseactivity.

DETAILED DESCRIPTION

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms include the pluralreference and vice versa unless the context clearly indicates otherwise.The term “or” is inclusive unless modified, for example, by “either.”Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about” “About” when used in connection with percentages means±1% unlessotherwise specified.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. The nomenclatures utilized in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

Disruption of microRNA (miRNA) gene regulatory pathways can lead tocancer. The paralogous RNA-binding proteins Lin28A and Lin28Bselectively block the expression of the let-7 family of miRNAs andfunction as oncogenes in a variety of human cancers. See U.S. PatentPub. No. 2010/0221266. For example, the majority of human colon andbreast tumors analyzed exclusively express either Lin28A or Lin28B:Lin28A is expressed in HER2-overexpressing breast tumors while Lin28Bexpression characterizes triple-negative breast tumors.

The present invention provides for compositions and methods formodulating the distinct mechanisms of Lin28A and Lin28B function. Asshown herein, Lin28B functions in the nucleus and inhibits theMicroprocessor by sequestering primary let-7 transcripts. Lin28Brepresses let-7 processing through a 3′ terminal uridylyl transferase(TUTase)-independent mechanism. In contrast, Lin28A-mediated inhibitionof let-7 expression involves recruitment of a TUTase (e.g.,Zcchc11/TUTase4/TUT4) to let-7 precursor RNA to block processing byDicer in the cell cytoplasm. As shown herein, biochemical dissection andreconstitution assays reveal the TUTase domains necessary and sufficientfor Lin28-enhanced pre-let-7 uridylation by the example TUTAse Zcchc11.In particular, a single C2H2-type zinc finger domain of Zcchc11 wasfound to be responsible for the functional interaction with Lin28. Lin28dramatically enhanced C2H2 binding to the terminal loop region ofpre-let-7. Additionally, Zcchc6 (TUTase7) acts as an alternative TUTasethat functions with Lin28 in vitro, and accordingly Zcchc11 and Zcchc6redundantly control let-7 biogenesis in embryonic stem cells.Importantly, the inhibitory effects of Zcchc11 depletion on thetumorigenic capacity and metastatic potential of human breast cancercells and xenografts is restricted to Lin28A-expressing tumors. Overall,the present invention provides insight into the mechanism ofLin28-mediated TUTase-dependent and Lin28B-mediated TUTase-independentcontrol of let-7 expression in development of stem cells and cancers. Asshown herein, Lin28A and Lin28B function by distinct mechanisms, whichhas implications for the development of new strategies for cancertherapy.

More specifically by way of introduction, control of gene expression bymicroRNAs (miRNAs) comprise a large family of regulatory RNAs that haveimportant roles in normal development. Zhao & Srivastava, 32 TrendsBiochem. Sci. 189 (2007). miRNAs are small, 22-nucleotide (nt) noncodingRNAs that repress the expression of many target messenger RNAs (mRNAs).Wightman et al., 75 Cell 855 (1993). Their requirement in mammals isdemonstrated by the severe developmental consequences of global loss ofmiRNAs, and more recently by the phenotypes observed in individual miRNAknockout mice. Hundreds of miRNAs have been identified, many of whichare developmentally regulated. Altered miRNA expression is linked withvarious diseases including cancer. Small & Olson, 469 Nature 336 (2011).

The canonical process of miRNA biogenesis is well understood and ischaracterized by successive cleavage events by RNase III enzymes. Winteret al., 11 Nat. Cell Biol. 228 (2009). The miRNA biogenesis pathwaybegins with transcription of primary miRNAs (pri-miRNAs); longtranscripts that contain a stem-loop structure with single-strandedflanking sequences. In the cell nucleus, pri-miRNAs are processed by theMicroprocessor protein complex, which contains an RNaseIII endonuclease,Drosha, and the double-stranded RNA-binding protein, DGCR8. TheMicroprocessor cleaves the double-stranded RNA towards the base of thestem-loop structure to release a 60-80 nucleotide (nt) hairpin-shapedprecursor (pre-miRNA) from the flanking RNA sequences. Denli et al., 432Nature 231 (2004); Gregory et al., 432 Nature 235 (2004); Han et al.,136 Cell 75 (2009). Pre-miRNAs are exported to the cell cytoplasm byExportin5, where they are subsequently cleaved by anotherdouble-stranded ribonuclease, Dicer. Hutvagner et al., 293 Science 834(2001); Krol et al., 11 Nat. Rev. Genet. 597 (2010). This cleavage stepgenerates a 22 nt miRNA duplex consisting of a guide and a passengerstrand. The guide strand is bound by Argonaute proteins and isincorporated into the RNA-induced silencing complex (RISC). Gregory etal., 123 Cell 631 (2005); Liu et al., 305 Science 1437 (2004).Base-pairing between the miRNA and the target mRNA guides the RISCcomplex to complementary transcripts leading to target gene repressionthrough mRNA degradation and/or translational repression. Krol et al.,2010. Proper temporal and spatial expression of miRNAs is essential fornormal development and physiology, as perturbations in specific miRNAsor miRNA processing factors can lead to aberrant development and cancer.Calin & Croce, 6 Nat. Rev. Cancer 857 (2006); Esquela-Kerscher & Slack,7 Cell Cycle 759 (2006); Small & Olson, 2011. Altered miRNA expressionis directly associated with cancer initiation, progression, andmetastasis and has been observed in a wide variety of humanmalignancies. Di Leva & Croce, 16 Trends 257 (2010).

The relationship between Lin28 proteins and the expression of miRNAlet-7 is central to the control of normal mammalian development, stemcell pluripotency, and cancer biology. Viswanathan & Daley, 140 Cell 445(2010). In embryonic cells, the RNA-binding protein Lin28 coordinatelyrepresses the let-7 family of miRNAs by binding to the terminal loop ofpre- and pri-let-7 miRNAs, thereby inhibiting let-7 biogenesis. Heo etal., 32 Mol. Cell 276 (2008); Newman et al., 14 RNA 1539 (2008); Rybaket al., 10 Nat. Cell Biol. 987 (2008); Viswanathan et al., 320 Science320 (2008). As cells undergo differentiation Lin28 levels decrease,leading to a corresponding increase in mature let-7, which is retainedin many adult tissues. As such, the posttranscriptional regulation oflet-7 expression by Lin28 contributes to the maintenance of thepluripotent state by preventing let-7 mediated ES cell differentiation.Martinez & Gregory, 7 Cell 31 (2010). Furthermore, Lin28 mRNA isrepressed by let-7 miRNAs, leading to an inversely correlated expressionpattern between let-7 and Lin28 and a double negative feedback loop thatcontrols cell differentiation. Wu & Belasco, 25 Mol. Cell Biol (2005).Lin28 is required for normal development and contributes to thepluripotent state by preventing let-7-mediated differentiation ofembryonic stem cells (ESCs). Ambros & Horvitz, 226 Science 409 (1984);Moss et al., 88 Cell 637 (1997); Viswanathan & Daley, 2010. Lin28overexpression or let-7 inhibition with antisense RNAs promotesreprogramming of human and mouse fibroblasts to induced pluripotent stemcells (iPSCs). Ambros & Horvitz, 1984; Melton et al., 463 Nature 621(2010); Yu et al., 318 Science 1917 (2007b).

The Lin28/let-7 axis is also relevant to a wide variety of human cancersas well as the control of glucose homeostasis in mammals. Frost & Olson,PNAS (2011); Iliopoulos et al., 139 Cell 693 (2009); Piskounova et al.,147 Cell 1066 (2011); Viswanathan et al., 41 nat. Genet. 843 (2009); Zhuet al., 147 Cell 81 (2011). In particular, the let-7 miRNA familymembers act as tumor suppressors in multiple different tumor types byinhibiting expression of oncogenes and key regulators of mitogenicpathways including RAS, MYC, and HMGA2. Bussing et al., 14 Trends Mol.Med. 400 (2008). Let-7 is downregulated in numerous different cancersand low let-7 correlates with poor prognosis. Boyerinas et al., 17Endrocr. Relat. Cancer F19 (2010); She et al., (2007); Takamizawa etal., 64 Cancer Res. 3753 (2004). Restoration of let-7 expression wasshown to effectively inhibit cancer growth in mouse models of lung andbreast cancers. Barh et al., 17 Curr. Oncol. 70 (2010); Esquela-Kerscheret al., 2008; Slack, 8 Cell Cycle 1823 (2009); Trang et al., 29 Oncol.1580 (2010); Yu et al., 131 Cell 1109 (2007a). In humans, there aretwelve let-7 family members (let-7a-1, -2, -3; let-7b; let-7c; let-7d;let-7e; let-7f-1, -2; let-7g; let-7i; miR-98) located at eight unlinkedchromosomal loci. Of note, many tumors are characterized by thecoordinate downregulation of all let-7 family members that are typicallyexpressed in the corresponding normal tissue. Shell et al., 104 PNAS11400 (2007). Previously, mechanisms controlling let-7 expressionremained largely unknown.

Further regarding Lin 28, there are two Lin28 paralogs in mammals:Lin28A (Lin28) and Lin28B. Guo et al., 384 Gene 51 (2006); Lehrbach etal., 16 Nat. Str. Mol. Biol. 1016 (2009); Moss et al., 1997; VanWynsberghe et al., 18 Nat. Str. Mol. Biol 302 (2011); Viswanathan &Daley, 2010. Lin28B has also been shown to regulate expression ofmultiple let-7 family members, and genome-wide association studies(GWAS) have linked Lin28B with the determination of human height, aswell as control of the age of onset of puberty and menopause. Lin28B(and less frequently Lin28A) contribute to oncogenesis by coordinatelyinactivating multiple let-7 family miRNAs. Iliopoulos et al., 2009;Viswanathan et al., 2009. This finding is consistent with the fact thatactivation of Lin28A/Lin28B occurs in many different primary humantumors with an overall frequency of −15% and these tumors displaydownregulation let-7 expression, suggesting an important role intumorigenesis. Indeed Lin28A/Lin28B are classical oncogenes that canpromote cellular transformation when ectopically expressed. Iliopouloset al., 2009; Viswanathan et al., 41 Nat. Genet. 843 (2009); West etal., 460 Nature 909 (2009). Importantly, this effect can be abrogatedwhen let-7 is reintroduced into these cells. Iliopoulos et al., 2009;Viswanathan et al., 2009. Therefore, Lin28-mediated cellulartransformation is directly dependent on let-7 levels.

Conversely, depletion of Lin28A or Lin28B in human cancer cells linesresults in decreased cell proliferation. Chang et al., 106 PNAS 3384(2009); Iliopoulos et al., 2009; Viswanathan et al., 2009. Lin28A/Lin28Bmay contribute to the development of an aggressive, poorlydifferentiated tumor. Lin28A or Lin28B expression is associated withadvanced disease in hepatocellular carcinoma (HCC), chronic myeloidleukemia (CML), Wilms' tumor, ovarian carcinoma, and germ cell tumors.Dangi-Garimella et al., 28 EMBO J. 347 (2009); Guo et al., 2006;Iliopoulos et al., 2009; Ji & Wang, 53 J. Hepatol. 974 (2010); Lu etal., 45 Eur. J. Cancer 2212 (2009); Oh et al. 76 Intl. J. Rad. Oncol.Biol. Phys. 5; Peng et al., 29 Oncol. 2153 (2010); Viswanathan et al.,2009; Wang et al., 31 Carcinogenesis 1516 (2010); West et al., 2009;Yang et al., 70 cancer Res. 9463 (2010). Lin28A/Lin28B expression isassociated with poor clinical outcome and patient survival in HCC andovarian cancer. Lu et al., 2009; Viswanathan et al., 2009. In the caseof LIN28B, rare amplification or translocation events might explainactivation in some cases. A more common mechanism, however, might betranscriptional activation by upstream factors during tumor progression.In support of this notion, c-Myc binds to both Lin28A and Lin28B lociand activates expression of these genes. Chang et al., 2009. Also, in abreast cancer model system, transient expression of Src oncoprotein inthe MCF10A cell line results in a transformed breast cancer cell linethat forms self-renewing mammospheres harboring tumor initiating cells.Iliopoulos et al., 2009. The transformation process involves NF-κBactivation leading to direct transcriptional upregulation of Lin28B,consequent let-7 loss, and de-repression of the let-7 target gene IL-6.Because IL-6 activates NF-κB, this regulatory circuit represents apositive feedback loop, providing a molecular link between inflammationand cancer.

Lin28A has been shown to recognize and selectively bind the terminalloop of let-7 precursors, a molecular recognition that requires both thecold-shock domain (CSD) and CCHC-type zinc finger RNA-binding domains ofthe Lin28A protein. Piskounova et al., 2008. Lin28A recognizes pre-let-7in the cytoplasm and recruits the terminal uridyl transferase (TUTase)Zcchc11 (TUTase4/TUT4) to add an oligouridine tail to the 3′ end ofpre-let-7, blocking Dicer cleavage and leading to the degradation of thepre-miRNA. Hagan et al., 16 Nat. Str. Mol. Biol. 1021 (2009); Heo etal., 2008; Heo et al., 138 Cell 696 (2009). Although both Lin28A andLin28B have been shown to have the ability to recruit Zcchc11/TUTase4 touridylate pre-let-7 in vitro, the molecular mechanism of theLin28B-mediated blockade of let-7 expression was unknown previously. Heoet al., 2008; Heo et al., 2009. The recently-identified TUTase Zcchc11may also regulate IL-6 levels by uridylating mature miR-26a, promote thecell-cycle-dependent degradation of a subset of histone mRNAs, and isrequired for the growth of Lin28A-driven cancers in vitro and in vivo.Jones et al., 11 Nat. Cell Biol 1157 (2009); Piskounova et al., 2011;Schmidt et al., 17 RNA 39 (2011). Given its central role in processesranging from the inflammatory response, to cell cycle regulation andLin28-mediated repression of let-7, TUTases, such as Zcchc11 and Zcchc6,are important RNA-modifying enzyme that may have essential roles indiverse aspects of human biology. The present invention characterizesthe molecular cis-acting elements of mammalian TUTases and how theseTUTases interact with their binding partners.

The present invention provides, surprisingly, that despite their highdegree of homology, Lin28A and Lin28B function through distinctmechanisms in human cancer cell lines. Lin28A and Lin28B aredifferentially localized in cells with Lin28A present predominantly inthe cell cytoplasm whereas Lin28B accumulates in the nucleus due to thepresence of functional nuclear localization signals in the Lin28Bprotein. Lin28B localizes to the nucleus where it binds pri-let-7 miRNAsto block processing by the Microprocessor through a TUTase-independentmechanism. In contrast, Lin28A functions in the cytoplasm by blocking atthe Dicer step and recruiting the TUTase to uridylate pre-let-7, aspreviously reported in mouse ES cells. Depletion of Zcchcl 1 has aneffect on let-7 expression only in Lin28A-expressing cancer cell lines,and Zcchcl 1 depletion selectively inhibits the tumorigenic capacity andmetastatic potential of Lin28A-expressing human breast cancer cells andxenografts. Because Zcchcl 1 has been shown to regulate let-7 it hasbeen identified as a potential therapeutic target in Lin28-expressingcancers. The present embodiments support the therapeutic potential ofZcchc11 inhibition in treating Lin28A-expressing cancers.

Nuclear Lin28B blocks processing of pri-let-7 miRNAs. Lin28A localizesto the cytoplasm and blocks Dicer processing of pre-let-7 microRNAs, byrecruiting Zcchc11. Lin28B localizes to the nucleus, where it blocks theMicroprocessor complex, possibly by binding and sequestering pri-let-7microRNAs in the nucleoli.

More specifically, the present invention provides for the mechanism bywhich Zcchc11 represses pre-let-7 in a Lin28-dependent manner.Mutational analyses of Zcchc11 identified domains required for activityboth in the absence and presence of Lin28, and use of recombinantproteins shows, herein, that Lin28 and Zcchc11 proteins are sufficientfor uridylation of pre-let-7 in vitro. Furthermore, the single C2H2-typezinc finger at the N-terminus of Zcchc11 mediates the interaction withLin28, and that this domain synergizes with Lin28 in binding topre-let-7. Comparing the domain architecture of Zcchc11 with othermammalian TUTases identified Zcchc6, another TUTase with extensivehomology to Zcchc11, that also mediates Lin28-dependent uridylation ofpre-let-7 in vitro. Accordingly, Zcchc6 depletion in embryonic cellssynergized with Zcchc11 knockdown to upregulate let-7 miRNAs, implyingthat these two TUTases work redundantly to repress let-7 expression.These findings provide insight into the mechanism of Lin28-mediatedTUTase control of let-7 expression in development, stem cells, andcancer.

As determined herein, Lin28B regulates let-7 expression through aZcchc11-independent mechanism. The paralogous RNA-binding proteinsLin28A and Lin28B have a high degree of sequence identity and conserveddomain organization (FIG. 1A) and both proteins selectively block let-7expression. See Newman et al., 2008; Viswanathan et al., 2008. Here,several human cancer cell lines were screened, revealing that someexpress Lin28A, whereas others express Lin28B (FIG. 1B). Theco-expression of both Lin28A and Lin28B was not observed in any cellline, suggesting that their expression may be mutually exclusive.Zcchc11 expression, however, was ubiquitous. More specifically, Helacells express Zcchc11 but neither Lin28A nor Lin28B. BecauseLin28A-mediated repression of let-7 in mouse ESCs involves the TUTaseZcchc11, whether Lin28A and Lin28B function through the same mechanismto block let-7 processing was determined. Previous reports have usedrecombinant Lin28A and Lin28B interchangeably in biochemical assaysdemonstrating that Lin28B is capable of enhancing Zcchc11 activity invitro, however the physiological relevance of these observationsremained unknown. Heo et al., 2009.

Whether both Lin28A and Lin28B function through a Zcchc11TUTase-dependent mechanism was investigated using co-immunoprecipitation(co-IP) experiments. Myc-tagged Lin28A, Lin28B, or Ago2 wereco-expressed with either Flag-tagged Zcchc11 or Flag-EIF6 control (FIG.1C). Because the Lin28A-Zcchc11 interaction has been shown to beRNA-dependent we also co-expressed pri-let-7g. Heo et al., 2009.Consistent with earlier reports, myc-Lin28A was found to be associatedwith affinity-purified Flag-Zcchc11. Heo et al., 2009. A physicalinteraction between myc-Lin28B and Flag-Zcchc11 was not detected.Additional co-IP experiments were performed in which the amount ofexogenously expressed Flag-Zcchc11 was titrated. These experimentsconfirmed the specific physical interaction Zcchc11 and Lin28A, whereasmyc-Lin28B was not detected in any of the Flag-Zcchc11 IPs (FIG. 2A).This was confirmed additionally by the co-IP of endogenous Lin28A inIgrov1 cells (FIG. 2B). Together, these results indicate that unlikeLin28A, there was no detectable physical interaction between Lin28B andZcchc11.

The functional requirement of Zcchc11 in the Lin28A- and Lin28B-mediatedrepression of let-7 expression was addressed using a series of knockdownexperiments to deplete Zcchc11 in a panel of human cancer cell lines.shRNAs were used to deplete Lin28A or Zcchc11 expression in Igrov cellsand measured the effect on let-7 expression by quantitative reversetranscription PCR (q.RT-PCR). As expected, depletion of Lin28A led to˜10-fold increase in let-7 levels. Knockdown of Zcchc11 with threeindependent shRNAs also led to elevated mature let-7 levels (FIG.1D-1G). Therefore Zcchc11 is involved in the repression of let-7expression in this Lin28A-expressing human cancer cell line as has beenpreviously reported in mESCs and P19 embryonal carcinoma cells. Hagan etal., 2009; Heo et al., 2009. Analogous experiments were performed inthree different Lin28B-expressing cancer cell lines: HepG2, K562 andH1299 (FIG. 1D-1G) and found no significant effect on mature let-7levels in any of the cell lines when Zcchc11 was depleted. In contrast,knockdown of Lin28B consistently led to the expected increase in maturelet-7. Overall our results indicate that Zcchc11 negatively regulateslet-7 expression in Lin28A- but not Lin28B-expressing cell linessuggesting that Lin28B employs a Zcchc11-independent mechanism to blocklet-7 processing.

Lin28B localizes to the nucleus, in contrast to Lin28A that is mostlypresent in the cell cytoplasm. Potential explanations for the functionaldifferences between Lin28A and Lin28B were examined, first, by comparingthe subcellular localization of the endogenous Lin28A with Lin28Bproteins using immunofluorescence assays (FIG. 3A). Lin28A was mostlylocalized to the cytoplasm of Igrov1 cells whereas Lin28B localized tospecific foci in the nucleus of H1299 cells where it co-localized withthe nucleolar marker Fibrillarin. Localization of Lin28B in the nucleoliwas confirmed by immunofluorescence assays on H1299 cells in whichLin28B expression (or control) was stably knocked down by shRNA andshowed that the observed nucleolar staining pattern is specific toLin28B (FIG. 3B). These data were further confirmed by biochemicalfractionation and Western blot of both cell lines (FIG. 3C). Consistentwith published data we found Zcchc11 only in the cytoplasmic fraction inboth the Lin28A- and Lin28B-expressing cell lines (FIG. 3C). These datasuggest that the divergence in the mechanisms by which Lin28A and Lin28Bblock let-7 biogenesis derives from their differential subcellularlocalization. The lack of physical and functional interactions betweenZcchc11 and Lin28B is therefore likely due to their localization todistinct cellular compartments, even though recombinant Lin28B has thecapacity to enhance Zcchc11 activity in vitro. Heo et al., 2009).

Lin28B contains functional nuclear localization signals. Lin28B proteinhas an extended C-terminus compared to Lin28A which upon closerinspection contains a putative bipartite nuclear localization signal(NLS), KK[GPSVQ]KRKK (SEQ ID NO:1). Another potential NLS,RRPK[GKTLQ]KRKPK (SEQ ID NO:2), was identified in the linker region thatconnects the two functional RNA-binding domains (FIG. 3D). To test thefunction of these putative NLS constructs for the expression of a seriesof GFP fusion proteins were generated. Hela cells were infectedtransiently with these constructs and analyzed the subcellularlocalization of the GFP-Fusion proteins by microscopy (FIG. 3E).Consistent with the localization of endogenous Lin28A in Igrov1 cells,we found Lin28A-GFP localized mainly to the cytoplasm. Lin28B-GFPpredominantly localized to specific foci in the nucleus, againrecapitulating the nucleolar localization of endogenous Lin28B observedin H1299 cells. Exogenously expressed truncated Lin28BΔNLS#1 increasedthe signal in the cytoplasm, however some nucleolar localization stillremained consistent with the presence of a second NLS. Indeed, thedouble mutant Lin28B-GFP protein lacking both NLS showed cellularlocalization similar to that of GFP alone suggesting that both NLSelements are important for nuclear and nucleolar localization of Lin28B(FIG. 3E). Whether these sequences represent functional NLS was examinedin the localization of the NLS#1-GFP and NLS#2-GFP (FIG. 3E). Whenexogenously expressed in Hela cells, NLS#1-GFP localized nearly entirelythroughout the nucleus including the nucleoli. This is in contrast tothe control GFP construct that is broadly distributed throughout thecell. Localization of NLS#2-GFP the signal was nearly entirely localizedto the nucleoli (FIG. 3E). Together these results identify that NLS#1amino acid sequence represents a bona fide NLS, and that NLS#2 is afunctional nucleolar localization signal (NoLS). NoLS properties areless well known and have only recently been studied at the amino acidsequence level. Scott et al., 38 Nucl. Acids Res. 7388 (2010). Severalreports suggest that proteins with a NoLS also contain an NLS thatallows them to cross the nuclear membrane before localizing to thenucleoli. These results were confirmed by biochemical fractionation ofHela cells transiently expressing either Flag-Lin28A, full-length-,truncated-, or double mutant-Flag-Lin28B. As expected Flag-Lin28A waspredominantly present in the cytoplasmic fraction, Flag-Lin28B wasmostly nuclear, NLS#1 mutant only showed marginal increase of signal inthe cytoplasmic fraction, whereas the double mutant Flag-Lin28B showed amore significant increase in cytoplasmic signal (FIG. 3F).

Lin28B directly associates with pri-let-7 miRNAs. To further dissect theexact mechanism of the Lin28B-mediated let-7 processing block, a stableFlag-Lin28B expressing HeLa cell line was created. Analysis of severalpri-miRNAs by q.RT-PCR in this cell line demonstrated a dramaticaccumulation of pri-let-7 miRNAs, over 10-fold for pri-let-7g and over3-fold for pri-let-7a-1. There was no effect, however, on levels ofpri-miR-21 (FIG. 4A). Consistent with this observation there was acorresponding decrease in the levels of mature let-7 miRNAs, with over90% decrease for mature let-7g, and about 40% decrease for maturelet-7a. Again no effect was observed on levels of mature miR-21 (FIG.4A). Also consistent with the Lin28B localization data, overexpressionof Lin28B in Hela cells causes a selective accumulation of pri-let-7miRNAs (and corresponding decrease in mature let-7 miRNA) presumably byblocking processing of pri-let-7 by the Microprocessor. Becausewild-type Hela cells do not express either Lin28A or Lin28B, pri-let-7levels were measured in the context of endogenous Lin28A- andLin28B-expressing cell lines. Indeed, the levels of pri-let-7g in aLin28A-expressing cell line, Igrov1, and two Lin28B-expressing celllines, H1229 and HepG2, showed higher levels of pri-let-7 miRNAs inLin28B-expressing cell lines compared with Igrov1 or Hela cells (FIG.4B). This is consistent with the notion that endogenous Lin28B blockslet-7 processing at the level of the Microprocessor leads toaccumulation of pri-let-7.

To gain further support for the model in which Lin28B functions in thecell nucleus to block the Microprocessor, the RNA that is associatedwith Lin28B protein was examined. Flag-Lin28A, Flag-Lin28B, andFlag-Lin28BΔNLS#1 were expressed individually in Hela cells and analyzedby Flag-immunoprecipitations. The RNA immunoprecipitation (RIP) wasperformed by extracting the RNA associated with the immunopurifiedproteins and analyzing relative levels of pri-let-7g by q.RT-PCR. Thisanalysis revealed that Lin28B directly associates with pri-let-7g RNA(FIG. 4C). Indeed, in comparison to mock IP, there was a −70-foldenrichment of pri-let-7 associated with Lin28B. Furthermore,substantially more pri-let-7 RNA was associated with Lin28B than withLin28A or with Lin28BΔNLS#1, which is consistent with the data showingdifferential localization of these proteins. As a control, theimmunopurified proteins were also used in an in vitro electromobilityshift assay (EMSA) to ensure that these Lin28 proteins had similarRNA-binding capacity. For this we performed EMSA with radiolabeledsynthetic pre-let-7a-1. Indeed purified Lin28A, Lin28B, and Lin28BΔNLS#1protein all displayed very similar binding affinity to pre-let-7 RNA(FIG. 4C). Taken together, these results indicate that the preferentialassociation of Lin28B with pri-let-7g detected in the RIP assays likelyreflects the distinct molecular mechanism of Lin28-mediated repressionof let-7 biogenesis and nuclear localization of Lin28B rather than anypossible intrinsic differential binding affinity to let-7 precursors.Therefore Lin28B, likely due to its nuclear localization, directlyassociates with pri-let-7 and corresponds with the accumulation ofpri-let-7 detected in Lin28B-expressing cells. This further supports themodel of the Lin28B-mediated block in let-7 biogenesis, in which Lin28Bblocks pri-let-7 processing by directly binding to the pri-let-7 andsequestering it from cleavage by the Microprocessor.

Additionally, Lin28B localizes to nucleoli where Microprocessor isabsent. The specific localization of Lin28B with that of the nuclearmiRNA processing machinery. The Microprocessor components, DGCR8 andDrosha, co-localize in the nucleoplasm but are excluded from nucleoli(FIG. 5A). To further confirm that localization of Lin28B is distinctand non-overlapping with the Microprocessor, the co-localization ofmCherry-DGCR8 and GFP-Lin28A/B proteins in transfected cells wasvisualized (FIG. 5B). Lin28A localized mostly to the cytoplasm andtherefore showed no overlap with the nuclear DGCR8. Lin28B localized tonucleoli and did not overlap with DGCR8 either. In contrast, thelocalization of the Lin28B NLS/NoLS mutant showed a broadly dispersedlocalization throughout the nucleus and cytoplasm (similar to GFPcontrol) and displayed co-localization with DGCR8 in the nucleoplasm.That Lin28B and Microprocessor normally occupy distinct compartments inthe nucleus was confirmed by performing large-scale biochemicalfractionation and Western blot of a stable Hela cell line expressingFlag-Lin28B. Lin28B was specifically present in the nucleolar-enrichedfractions whereas DGCR8 was only detectable in the nuclear fraction andnot in the nucleolar fractions (FIG. 5C). Overall, these findingssuggest a possible mechanism by which Lin28B blocks let-7 processing inthe nucleus by sequestering pri-let-7 miRNAs in the nucleoli away fromthe Microprocessor.

More specifically, that Lin28B blocks processing of pri-let-7 miRNAs bythe Microprocessor was confirmed using a novel dual luciferase reportersystem to monitor Microprocessor activity in transfected cells (FIG.6A). A psiCHECK2 vector was used and several reporter constructsgenerated by placing particular pri-miRNA sequences between the RenillaLuciferase open reading frame and the polyA site. This novel reporterallows monitoring of Microprocessor activity in vivo. In the presence ofan active Microprocessor complex the pri-miRNA should be processedregularly and Renilla protein expression would be consequently low.Inhibition of the Microprocessor should lead to stabilization of theRenilla mRNA and thereby lead to accumulation of Renilla protein. TheRenilla Luciferase signal can be normalized to the Firefly luciferasethat is encoded in the same plasmid but expressed ubiquitouslyregardless of Microprocessor activity. To validate this system as areliable sensor of Microprocessor activity, DGCR8 levels were depletedby transient knockdown with siRNAs in Hela cells, which co-expressed thedual luciferase plasmid (FIG. 6B). A scrambled siRNA was used as anegative control. Three different dual luciferase constructs were usedcontaining pri-let-7g, pri-let-7a-1, or pri-miR-125 (FIG. 6C). Thisshowed that for all three constructs, Renilla Luciferase was stabilized,when DGCR8 was depleted. As a control for the luciferase system we usedan empty vector with no pri-miRNA sequence, and showed that the relativeLuciferase levels were unchanged by DGCR8 depletion (FIG. 6C). As thenext step to address the question of whether Lin28B directly inhibitsthe action of the Microprocessor, Lin28B was transiently overexpressedin Hela cells in the presence the dual luciferase plasmids containingthe different pri-miRNAs. The results demonstrate that overexpression ofLin28B caused stabilization of both pri-let-7 reporters, but had noeffect on the pri-miR-125 construct (FIG. 6D). This result furthersupports the hypothesis that Lin28B inhibits the action of theMicroprocessor in the nucleus. To confirm that the same mechanism isfunctional in cell lines that express endogenous Lin28B, was confirmedusing the dual luciferase system in Lin28A- and Lin28B-expressing celllines. Consistent with the observations for exogenously expressedLin28B, pri-let-7 constructs were more stable in Lin28B-expressing celllines than in Lin28A expressing cells (FIG. 6E). This confirmed thatendogenous Lin28B operates via a mechanism distinct to that of Lin28A,and blocks let-7 processing by inhibiting the action of theMicroprocessor complex.

Furthermore, Lin28B directly binds and sequesters pri-let-7. To furtherdissect the mechanism of the Lin28B-mediated let-7 processing block, therelative abilities of recombinant human Lin28A and Lin28B proteins tobind pre-let-7 were compared. (FIG. 7A, 7B and FIG. 8). EMSA wasperformed with pre-let-7g to analyze the relative binding affinities ofthe two recombinant proteins. Lin28A and Lin28B have apparent Kds ofapproximately 0.6 nM and 0.5 nM, respectively (FIG. 7B). Both theseestimated Kds are much lower than a previously reported for recombinantmouse Lin28A. This difference is likely due to the omission here ofnonspecific yeast tRNA competitor used previously in the binding buffer.Piskounova et al., 2008. An EMSA with pri-let-7g was performed anddemonstrated that both recombinant Lin28A and Lin28B are able to bindpri-let-7g with similar affinities (FIG. 7C). Collectively, these assaysreveal that both Lin28 proteins can directly bind to let-7 precursorswith high-affinity in vitro.

To gain further support for our model in which Lin28B binds andsequesters pri-let-7 in the nucleus to inhibit the Microprocessor, theRNA associated with Lin28B was examined. Flag-Lin28A and Flag-Lin28Bwere individually expressed and purified, and the associated RNAextracted and analyzed relative levels of pri-let-7g by q.RT-PCR. ThisRNA immune-precipitation (RIP) analysis revealed that Lin28B directlyassociates with pri-let-7g RNA (FIG. 7D), revealing an ˜18-foldenrichment of pri-let-7 associated with Lin28B. Furthermore,substantially more pri-let-7 associated with Lin28B than with Lin28A,which is consistent with the differential subcellular localization ofthese proteins. Taken together, these results indicate that thispreferential association of pri-let-7g with Lin28B likely reflects thedistinct mechanism by which Lin28B represses let-7 expression ratherthan any possible intrinsic differences in the relative RNA-bindingaffinities of Lin28A and Lin28B proteins.

The effect of transient Lin28B overexpression on pri-let-7g levels wasalso studied by q.RT-PCR. Transient Lin28B overexpression led to˜12-fold accumulation of pri-let-7g levels (FIG. 7E). In contrast,overexpression of Lin28A only had a more modest effect on pri-let-7glevels consistent with its predominantly cytoplasmic localization. Inorder to further assess the effects of Lin28B overexpression on bothpri- and mature miRNA levels, a Flag-Lin28B expressing Hela stable cellline was used. Analysis of several pri-miRNAs by q.RT-PCR in this cellline demonstrated a substantial accumulation of pri-let-7miRNAs, >10-fold for pri-let-7g and >3-fold for pri-let-7a-1. There washowever no effect on levels of pri-miR-21 (FIG. 4A). A correspondingdecrease in the levels of mature let-7, with >90% decrease for maturelet-7g, and ˜40% decrease for mature let-7a was observed. Again noeffect was observed on levels of mature miR-21 (FIG. 4A). Together,these data support our model whereby nuclear Lin28B directly associateswith pri-let-7 sequestering it from cleavage by the Microprocessor toselectively inhibit let-7 maturation, and underscore our findings thatthe paralogous RNA-binding proteins, Lin28A and Lin28B, operate bydistinct mechanisms to selectively repress let-7 expression.

Zcchc11 inhibition blocks the tumorigenicity and invasiveness of Lin28A-but not Lin28B-expressing breast cancer cells in vitro and in vivo. Themechanistic studies showed that Lin28A and Lin28B block let-7 processingthrough distinct mechanisms: Lin28A recruits Zcchc11 to uridylatepre-let-7 miRNAs in the cytoplasm, whereas Lin28B functions in thenucleus through a Zcchc11-independent mechanism by blocking pri-let-7biogenesis at the Microprocessor step. Next, the physiological anddisease relevance of these distinct mechanisms were explored in thecontext of the role of Zcchc11 in human Lin28A- and Lin28B-expressingcancer, more specifically, the effect of Zcchc11 inhibition on thetumorigenicity and invasiveness of breast cancer cells expressing eitherLin28A or Lin28B.

Initially, the effects of Zcchc11 inhibition in MCF10A ER-Src induciblemodel of cellular transformation were tested, where MCF10A immortalizedbreast epithelial cells become transformed 36 h post tamoxifen (TAM)treatment. Iliopoulos et al., 2009. During transformation of MCF10AER-Src cells, there is activation of a negative feedback loop,consisting of NF-κB, Lin28B, let-7a and 1L6, which is essential forinduction and maintenance of the transformed phenotype. During thetransformation process, there is an increase in the expression levels ofLin28B, while these cells do not express Lin28A (FIG. 9A). Furthermore,when the MCF10A ER-Src cells are transformed (36 h post TAM treatment)they have a substantial change in their morphology (FIG. 9B). Zcchc11depletion by siRNA did not affect MCF10A ER-Src transformation, while incontrast depletion of Lin28B by siRNA or inhibition of IL6 by amonoclonal antibody (Ab-IL6) suppressed this transformation. Inaddition, in order to confirm these findings, we tested the effects ofZcchc11 inhibition on the colony formation ability of the transformedMCF10A ER-Src cells. Interestingly, Zcchcl 1 suppression did not affectthe colony formation ability of the transformed MCF10A ER-Src cells,while inhibition of Lin28B blocked their tumorigenicity (FIG. 9C, 9D).Furthermore, Zcchc11 inhibition did not affect the expression levels oflet-7a miRNA and its direct downstream target 1L6 (FIG. 9D, 9E, 9F).

In addition to the in vitro data, the effects of Zcchc11 inhibition inwas tested in xenografts (FIG. 9G). Specifically, MCF10A ER-Srctransformed cells were injected in immunodeficient (nu/nu) mice and whenthe tumors reached the size of 100 mm3, the mice were randomlydistributed into four groups (five mice/group). The first group of micedid not receive any treatment (untreated), the second group of mice wastreated intraperitoneously (i.p.) with siRNA negative control (siRNA NC)the third group of mice was treated i.p. with siRNA against Zcchc11(siZcchc11#1) and the fourth group of mice was treated i.p. with siRNAagainst Lin28B (siLin28B). All the treatments started at day 15 for 5cycles (days 15, 20, 25, 30, 35). In accordance with the in vitro data,Zcchc11 inhibition did not affect MCF10A ER-Src tumor growth, whileLin28B inhibition suppressed tumor growth (FIG. 9G, 9H, 9I). The tumorswere excised from the mice at day 30 (after three cycles of treatment)and were tested for let-7a expression. siZcchc11 treatment did notaffect let-7a expression levels in these tumors, while siLin28Btreatment led to a 7-fold increase in let-7a expression levels (FIG.9J). Overall, these data suggest that inhibition of Zcchc11 does nothave an inhibitory effect on the tumorigenicity and invasiveness ofLin28B-expressing MCF10A ER-Src cells.

To further explore the distinct requirements for Zcchc11 in Lin28A- andLin28B-expressing cancer, the effects of Zcchc11 inhibition on thetumorigenicity and invasiveness of MDA-MB-231 breast cancer cells(Lin28B-expressing cells) relative to T47D breast cancer cells(Lin28A-expressing cells) were compared. Suppression of Zcchc11expression did not affect let-7a expression in MDA-MB-231 cells, but ledto 7-fold increase in mature let-7a levels in T47D cells (FIG. 10A,10B). Furthermore, Zcchc11 inhibition did not affect the tumorigenicityand invasiveness of MDA-MB-231 cells, while it suppressed both thecolony formation ability and invasiveness of T47D cells (FIGS. 10C,10D). Zcchc11 inhibition had similar effects on the tumor growth ofthese cell lines in xenografts. Specifically, Zcchc11 knockdown did notaffect tumor growth of MDA-MB-231 while it suppressed T47D tumor growth(FIG. 10E). Synthetic let-7a miRNA suppressed both MDA-MB-231 and T47Dtumor growth (FIG. 10E). Also, in the tumors derived from MDA-MB-231xenografts (day 30), let-7a expression was not affected by inhibition ofZcchc11, while Lin28B suppression increased let-7a levels about 5-fold(FIGS. 5F, 5F, 10F, and 10G). On the other hand, both Zcchc11 and Lin28Ainhibition resulted in up-regulation of let-7a expression to similarlevels in T47D-derived tumors (day 30) (FIG. 10F, 10G).

In addition to the breast cancer cells, the effects of Zcchc11inhibition on tumor growth of several other (liver, lung, ovarian,melanoma, colon) cancer cell types were tested (FIGS. 11A-11B). Zcchc11inhibition (FIG. 11C) blocked the growth of LIN28A-expressing tumors(Igrov1) and did not affect the growth of Lin28B-expressing tumors(HepG2, H1299, SK_MEL_28, CaCO2) (FIG. 11A-11B). Lin28A and Lin28Binhibition suppressed the growth of the corresponding tumors (FIGS.11D-11E). Taken together, these data suggest that Zcchc11 plays a rolein the tumorigenicity and invasiveness of Lin28A-expressing cancer cellsbut depletion of Zcchc11 in Lin28B-expressing cancer cell lines has noeffect on cancer growth.

The disease relevance of these findings were further confirmed bymeasuring Lin28A and Lin28B expression in human colon and breasttissues. Lin28A or Lin28B are upregulated while let-7a was downregulatedin colon adenocarcinomas relative to normal colon tissues (FIG. 12A).Specifically, Lin28A was upregulated in 19/45 colon adenocarcinomaswhile Lin28B was upregulated in 14/45 colon adenocarcinomas. The colontumors with Lin28A overexpression had very low levels of Lin28B and viceversa. Furthermore, immunohistochemistry and in situ hybridizationanalyses in normal and colon cancer tissues revealed that Lin28A orLin28B proteins were upregulated while let-7a was downregulated in coloncarcinomas relative to normal colon tissues (FIG. 12B, FIG. 13). Similarto the mRNA data, immunohistochemistry revealed that tumors thatexpressed high Lin28A protein levels had low levels for Lin28B and viceversa. This is consistent with our analysis of human cancer cell lineswhere we did not find cells that express both Lin28A and Lin28B (FIG.1B).

Additionally, Lin28A or Lin28B were upregulated while let-7a wasdown-regulated in breast cancer relative to normal breast tissues (FIG.12C). Specifically, Lin28A was upregulated in 9/33 breast carcinomaswhile Lin28B was upregulated in 10/33 breast carcinomas. Similar to thecolon tissues, the breast tumors that overexpressed Lin28A had very lowlevels of Lin28B and vice versa. In addition, Lin28A was significantlyupregulated in HER2-overexpressing breast tumors while Lin28B wassignificantly upregulated in triple-negative (ER-,PR-,HER2-) breasttumors (FIG. 12D). Furthermore, according to previous studies Lin28Bexpression is a part of an inflammatory circuit and is regulated byNF-κB transcription factor. Iliopoulos et al., Cell, 2009. Thus, howNF-κB activity correlates with Lin28A or Lin28B mRNA levels in humanbreast tumors was tested, and revealed a statistically significantcorrelation between NF-κB nuclear levels and Lin28B but not Lin28Aexpression levels (FIG. 12E). These data suggest that NF-κB regulatesLin28B but not Lin28A pathway. Also, in order to have a broader view ofLin28A and Lin28B expression levels in human cancer tissues, theirexpression levels in kidney, liver, lung, ovarian, prostate, and thyroidcancer were compared (FIG. 12F). These data reveal that Lin28B isupregulated in liver, ovarian and thyroid carcinomas.

The present invention also provides the domains of Zcchc11 required forLin28-enhanced pre-miRNA uridylation. As described herein and elsewhere,Zcchc11 is a cytoplasmic Lin28-interacting TUTase in embryonic andcancer cells. Hagan et al., 2009; Heo et al., 2009; Piskounova et al.,2011. Its depletion in Lin28-expressing cells leads to the specificupregulation of let-7 family members similar to the depletion of Lin28and its expression is required for potent let-7 repression and rapidcell growth in Lin28A-expressing cancers. Piskounova et al., 2011.Zcchc11 encodes a 184 kDa non-canonical poly(A) polymerase that ishighly conserved across vertebrates. The Zcchc11 active site is locatedwithin the Nucleotidyl Transferase (Ntr) domain, which is paired with aPoly(A)-Polymerase-Associated (PAP) domain; a common feature ofnon-canonical poly(A) polymerases. Kwak & Wickens, 13 RNA 860 (2007);Martin & Keller, 7 Stem Cell 31 (2007); Saitoh et al., 109 Cell 563(2002). Catalysis requires a conserved Aspartate triad in the Ntr, andan overexpressed mutant lacking these residues functions as a dominantnegative. Hagan et al., 2009. Flanking the active site are three CCHCretroviral-type zinc fingers/zinc knuckles, which are implicated innucleic acid binding. At the N-terminus of the protein is a region thatshares significant homology with the active site, including a proximalPAP domain; however, this region lacks one of the crucial Aspartatespredicted to be necessary for catalysis. Instead, this region is mostsimilar to the yeast TRF4 proteins, which carry out cytoplasmic poly(A)RNA polymerase activity. Saitoh et al., 2002. N-terminal to this regionis a classical C2H2 zinc finger with no known function. These motifs areknown to bind DNA, RNA, or protein. Finally, at the N- and C-termini ofZcchc11 there are two domains of unknown function similar, respectively,to pneumoviridae attachment proteins and the glutamine-richneurodegenerative disease-associated protein atrophin-1 (FIG. 14).

To understand which domains of Zcchc11 are required for uridylationactivity, a series of mutant cDNAs were generated and tested the abilityof the resulting Flag-immunopurified (Flag IP) proteins to uridylatesynthetic pre-let-7 miRNA in vitro. Mutants were generated lacking N-and C-terminal domains or harboring point mutations in conservedresidues (FIG. 14A). As described previously, Zcchc11 exhibits alow-level of uridylation against pre-let-7 and this activity is stronglyenhanced by the addition of recombinant or immunopurified Lin28. Haganet al., 2009; Heo et al., 2009. As determined by the incorporation ofradiolabeled UTP, the activity of wild type Zcchc11 was compared to eachof the series of mutants (FIG. 14B). The serial N-terminal truncationsshowed that the pneumoviridae (PneumoG) domain was dispensable for bothbasal level uridylation and activity enhanced by IP Flag-Lin28. Loss ofthe N-terminal C2H2 domain still allowed for basal activity, but thismutant could no longer support Lin28-enhanced uridylation againstpre-let-7, indicating that the C2H2 zinc finger may be essential for theinteraction between Lin28 and Zcchc11. Interestingly, when the TRF4domain was deleted, neither basal nor Lin28-enhanced activities weredetected. This result was surprising given the prediction that the TRF4domain was insufficient to carry out catalytic activity on its own.Indeed, when a fragment of Zcchc11 containing the N-terminal TRF4 domainbut lacking the NTR domain was tested, no uridylation activity wasdetected (FIG. 14B, upper panel, compare lanes 7 and 8 to 11 and 12).All further mutant proteins tested lacking TRF4 failed to support anydetectable uridylation activity (FIG. 14B, upper panel, lanes 9 and 10).

To determine if the findings on N-terminal deletions of Zcchc11 could besupported in the context of additional C-terminal truncations, mutantslacking the C-terminal Atrophin-like domain in combination with ΔPneumoGand ΔC2H2 mutants were tested. The Atrophin-like domain was dispensablein these experiments, indicating that it is not required for basal orLin28-enhanced uridylation by Zcchc11. To confirm that the C2H2 zincfinger per se was required for Lin28-enhanced uridylation, a full-lengthZcchc11 cDNA was generated bearing Cysteine to Alanine mutations in theresidues predicted to be central to the C2H2 zinc finger (C326/329A).Indeed, this mutant exhibited only basal uridylation activity, as theaddition of Lin28 had no impact on its catalysis in vitro. Given thatthis mutant phenocopied the ΔC2H2 and ΔC2H2/C mutants, we conclude thatthis zinc finger is required for Lin28-enhanced uridylation in vitro.

To define the minimal Zcchc11 mutant that supports Lin28-enhanceduridylation, the requirements of C-terminal domains were examinedfurther. Compared to WT, a mutant lacking the C-terminal-most CCHC zincfinger exhibited robust basal and Lin28-enhanced activity, whereasadditionally truncating the adjacent CCHC zinc finger led to nodetectable activity, implying that the three CCHC zinc fingers may berequired for different aspects of RNA recognition or positioning (FIG.14B, lower panel). These studies provide insight into the basicmechanism underlying the catalytic nature of Zcchc11 (FIG. 14C).

In vitro reconstitution of Lin28-mediated pre-let-7 uridylation wasachieved with recombinant proteins. The experiments described hereinsuggest that specific domains of Zcchc11 mediate the interaction withLin28 to uridylate pre-let-7 in vitro. To confirm that these twoproteins are sufficient for activity and do not rely on contaminating oraccessory factors interacting with the immunopurified proteins, 6×-HisLin28 (r.Lin28) and Flag/6×-His ΔPneumo G/C Zcchc11 were expressed in apurified from E. coli. Compared with immunopurified ΔPneumo G/C Zcchc11,the Flag/6×-His protein uridylated pre-let-7 at the basal level to asimilar extent indicating that the Zcchc11 expressed and purified frombacteria is catalytically active. Adding either immunopurifiedFlag-Lin28 or recombinant 6×-His Lin28 to the reaction similarlyenhanced the uridylation of pre-let-7 by either Zcchc11 preparation.Neither of the Lin28 proteins themselves led to detectable levels ofuridylated pre-let-7, indicating that labeled products originated fromthe enzymatic activity of Zcchc11. These experiments show that thecombination of Lin28 and Zcchc11 proteins are necessary and sufficientto carry out the robust uridylation of pre-let-7 in vitro.

The C2H2 zinc finger of Zcchc11 synergizes with Lin28 to bind pre-let-7.The discovery that Zcchc11 requires its N-terminal C2H2 zinc finger tomediate Lin28-enhanced uridylation suggested that this region isimportant for physically interacting with either Lin28 or recognizing aLin28-bound form of pre-let-7. This was tested using electrophoreticmobility shift assays (EMSA) with recombinant 6×-His Lin28 and afragment of the N-terminus of Zcchc11 containing the C2H2 zinc finger(r.C2H2) (FIG. 16A). As shown previously, incubating r.Lin28 with 5′end-labeled pre-let-7 leads to the formation of a stable complex thatcan be resolved on a native polyacrylamide gel (Piskounova et al., 2008)(FIG. 16B, lane 2). Compared with r.Lin28, r.C2H2 bound poorly topre-let-7g alone (FIG. 14B, lanes 5 and 6). When both proteins werecombined, however, the intensity of the bound probe was greater than theadditive effect of the two proteins singly bound to the probe (FIG. 14B,compare lane 4 to lanes 2 and 6). Although this binding synergy dependedon both proteins, there is no detectable ternary complex formation,suggesting that the bound probe is interacting with only one of the twoproteins. To further investigate the binding dynamics of Lin28 and theC2H2 zinc finger of Zcchc11, EMSA experiments were conducted using onlythe terminal loop region of pre-let-7 that is removed by Dicer, morerecently termed the pre-element or preE. Nam et al., 147 Cell 1080(2011); Piskounova et al., 283 J. Biol Chem. 21310 (2008).

Let-7 preE is sufficient for Lin28 binding and this binding requires RNAsequence and structural information encoded in the preE. Heo et al.,2009; Nam et al., 2011; Newman et al., 2008; Piskounova et al., 2008. Toexamine more sensitively the relative affinities of both r.Lin28 andr.C2H2, EMSA experiments were carried out at sub-saturating conditionsusing the preE of let-7g. As shown in FIG. 16C, weak binding of eitherr.Lin28 or r.C2H2 could be dramatically enhanced by the addition of bothproteins (compare lanes 3 and 5 to lane 9). Again, only a single bandwas observed, indicating a complex composed of pre-let-7 and one of therecombinant proteins. These experiments demonstrate that althoughbinding of either protein can occur, the presence of the second proteinincreases the affinity for let-7-derived RNAs.

Although the conformational changes underlying this effect have not beendetailed, structural studies and RNA footprinting experiments haverevealed changes in the preE of various let-7 family members uponbinding by Lin28. Desjardins et al., Nucl. Acids Res. (2011); Lightfootet al., 50 Biochem. 7514 (2011). This change in RNA structure uponbinding by Lin28 may allow for specific protein-RNA and/orprotein-protein interactions that further alter the RNA or proteinstructure and permit an increase in C2H2 affinity for the RNA. Theseresults echo the findings from in vitro uridylation assays where Zcchc11activity is low when incubated with pre-let-7 alone, but dramaticallyincreases in the presence of Lin28. Synergistic binding between Lin28and the C2H2 zinc finger of Zcchc11 provides greater insight into themechanism by which the let-7 degradation pathway is controlled andprovides further support for the requirement of the C2H2 domain forLin28-enhanced pre-miRNA uridylation (FIGS. 14A-14C).

Additionally, the let-7 preE confers Lin28-enhanced pre-miRNAuridylation by Zcchc11. To understand the role of pre-miRNA substratesin Zcchc11-mediated uridylation, which cis-acting RNA elements supporturidylation enhanced by Lin28 were explored. Lin28 binding to pre-let-7requires specific sequence and structural information in both the RNAand the protein. The cold-shock domain (CSD) of Lin28 is inserted intothe terminal loop of various pre-let-7 RNAs, and the Lin28 CCHC zincfingers dimerize to recognize a GGAG motif proximal to the Dicercleavage site of pre-let-7. Loughlin et al., 2011; Nam et al., 2011.

The regions of pre-let-7 required for uridylation by Zcchc11 wereidentified in the context of the understanding that the let-7 preE isbound by recombinant Lin28 as efficiently as full-length pre-let-7.Piskounova et al., 2008. If Lin28 binding is sufficient to directZcchc11-mediated uridylation, then RNA substrates with divergentsequences outside of the let-7 preE should be comparable substrates topre-let-7. To test this, a synthetic pre-miRNA was generated composed ofthe preE of let-7g and the stem sequence of miR-21 (pre-21S7L) (FIG.17A), and compared the uridylation activity of Zcchc11 towards this RNAversus both pre-let-7g and pre-miR-21. As shown in FIG. 17B, pre-let-7gundergoes robust uridylation with the addition of IP Flag-Lin28. miR-21is uridylated at a basal level similar to that of pre-let-7, but theaddition of IP Flag-Lin28 has no effect on uridylation levels asdescribed previously. Hagan et al., 2009. When the chimeric pre-S21L7 isincubated with IP Flag-Lin28, however, it is subjected to enhanceduridylation activity similar to that of WT pre-let-7 (FIG. 17B, comparelanes 5 and 6 to 1 and 2). This result suggests that the effect of Lin28binding to the preE of pre-let-7 is sufficient to allow targeting anduridylation by Zcchc11.

A related TUTase Zcchc6 is functionally redundant with Zcchc11 in vitro.The findings on the domains of Zcchc11 supporting Lin28-mediateduridylation in vitro led to the examination of other TUTases aspotential regulators of pre-miRNAs. Among the seven non-canonicalpoly(A) polymerases encoded in the human genome, Zcchc6 (PAPD6/TUTase 7)has striking homology to Zcchc11 including the domains constituting itsactive site, its three CCHC zinc fingers, the N-terminalTRF4/PAP-associated domains, and C2H2 zinc finger (FIG. 18A).Importantly, there is extensive conservation between Zcchc11 and Zcchc6at critical residues in the active site and in the C2H2 zinc finger(FIGS. 18A-18C). Whether Zcchc6 shares activity similar to Zcchc11 couldbe seen by the ability of IP Flag-hZcchc6 to uridylate pre-let-7 invitro in the absence or presence of Lin28. Similar amounts ofFlag-hZcchc11 or Flag-hZcchc6 were used in uridylation assays withFlag-hLin28A and both TUTases were stimulated to an equal extent (FIGS.19A, 19C). To confirm that the effects seen with Flag-hLin28A were notdependent on the paralog of Lin28 used, the stimulatory effect ofFlag-hLin28B was tested, because both Lin28 proteins act identically invitro (FIGS. 19A-19C). Heo et al., 2009. In these experiments eitherLin28A or Lin28B enhanced the uridylation activity of either TUTase in adose-dependent manner. The enhancement in hZcchc6 uridylation activitywas also observed using r.Lin28, indicating that the this effect was notdue to coimmunoprecipating proteins and that Zcchc6 and Zchc11 arefunctionally indistinguishable in these assays (FIG. 19D).

These in vitro results suggest both TUTases may recognize let-7precursors in biologically relevant settings. Zcchc6 has previously beenshown to have poly(U) activity in vitro (Kwak & Wickens, 2007; Risslandet al., 27 Mol. Cell. Biol. 3612 (2007)), and depletion of Zcchc6 incolon cancer cells led to reduced levels of uridylated mature let-7e.Wyman et al., 21 Genome Res. 1450 (2011). Zcchc6 is also a homolog of C.elegans CDE-1, which uridylates a subset of siRNAs bound by theArgonaute protein CSR-1 and loss of CDE-1 leads to aberrant chromosomalsegregation and dysregulation of CSR-1-bound siRNAs. van Wolfswinkel etal., 139 Cell 135 (2009). In spite of these data, this is the firstevidence of Zcchc6 uridylating pre-miRNAs and suggests parallel activitywith Zcchc11 and a role in the Lin28 pathway.

The present invention also demonstrates that Zcchc11 and Zcchc6redundantly control let-7 biogenesis in embryonic stem cells. Given thefindings on the activity of Zcchc6 in vitro, whether Zcchc6 functions inparallel with Zcchc11 in vivo was explored. Zcchc11 depletion inembryonic carcinoma (EC) and embryonic stem (ES) cells led to thecoordinate derepression of let-7 miRNAs, while Zcchc6 depletion led tono change in mature let-7 levels. Hagan et al., 2009; Heo et al., 2009.The derepression observed upon Zcchc11 knockdown was generally moremodest, however, than the depletion of Lin28 in all cell types tested.Hagan et al., 2009; Heo et al., 2009; Piskounova et al., 2011. Oneinterpretation of this finding is that there are redundant factorsworking in parallel with Lin28 and Zcchc11 to repress let-7 miRNAs inundifferentiated cell types. Whether Zcchc6 works redundantly withZcchc11 was tested using siRNAs to deplete both TUTases in P19 and V6.5cell lines (EC and ES cells, respectively). Upon Zcchc11 knockdown withtwo independent siRNAs, there was a modest 2- to 3-fold upregulation ofmature let-7g, whereas consistent with previous reports, depletion ofZcchc6 with two independent siRNAs led to no significant changes at thelevel of mature let-7g. When both TUTases were knocked down, however,there was a consistent upregulation in mature let-7 that was moredramatic than either individual knockdown alone (FIG. 20). This trendwas specific to let-7 family members, as levels of the unrelated miRNAmiR-21 were unchanged. Moreover global profiling revealed that changesin miRNA expression were restricted to let-7 family members. This trendwas seen in both P19s and V6.5s, suggesting Zcchc11/Zcchc6 redundancy isa general mechanism of embryonic cells. The synergistic relationshipbetween these two related TUTases, both in vitro and in vivo, couldexplain the modest effects seen for depletion of either TUTaseindividually in Lin28-expressing cells and expands the repertoire ofmiRNA modifying enzymes.

Prior to the instant invention, because Lin28A and Lin28B inhibit let-7miRNA biogenesis in ESCs and cancer it had been assumed that bothproteins block let-7 expression through the same mechanism, byrecruiting the TUTase Zcchc11 (TUT4) in the cell cytoplasm to uridylatepre-let-7 and target it for degradation. Moreover these paralogousproteins have been used interchangeably in several in vitro assays. Heoet al., 2009. The present results provide the first evidence thatdespite their high degree of homology, Lin28A and Lin28B functionthrough distinct mechanisms to block let-7 processing, a finding thathas broad implications for the development of new cancer therapies andthe potential use of Zcchc11 inhibitors in Lin28A-expressing tumors butnot in Lin28B-expressing tumors. This distinction derives from thedifferential subcellular localization of these two proteins: Lin28Alocalizes primarily to the cytoplasm, whereas Lin28B contains functionalnuclear localization signals and specifically localizes to nucleoli.

Due to the differential subcellular localization of the two proteins, inhuman cancer cell lines, Lin28A and Lin28B block let-7 processing atdifferent steps of the miRNA-processing pathway. The steps at whichlet-7 processing is blocked by Lin28 in various different organisms iscontroversial, however. A recently published report proposes that Lin28binds pri-let-7 and blocks let-7 expression co-transcriptionally in C.elegans and disputes earlier conclusions that Lin28 functions at stepsfurther downstream in the let-7 biogenesis pathway. Lehrbach et al.,2009); Van Wynsberghe et al., 2011. Others also report that a very smallfraction of Lin28A in hESCs localizes to the nucleus and binds pri-let-7miRNAs, although pre-let-7 is bound abundantly. Van Wynsberghe et al.,2011. This is consistent with the results that demonstrate a smallfraction of Lin28A localizes to the nucleus in Igrov1 cell line. Also,Lin28A binds pri-let-7 miRNA, however not as much as Lin28B, and earlierreports have demonstrated that purified Lin28A can inhibit theMicroprocessor in vitro. Newman et al., 2008; Viswanathan et al., 2008.Additionally, although the data presented herein demonstrate thatLin28B-mediated repression of let-7 expression is Zcchc11(TUT4)-independent in multiple different cell-types, it remains possiblethat in certain contexts or cell-types Lin28B may localize to thecytoplasm and utilize Zcchc11/TUT4 to repress let-7 biogenesis. Forexample, uridylated pre-let-7 was previously detected in Huh7 cells andLin28B is reportedly localized to the cytoplasm in Huh7 cells. Guo etal., 2006; Heo et al., 2008.

The present demonstration that the Microprocessor is excluded fromnucleoli raises the possibility that sequestration of certain pri-miRNAsto nucleoli by specific RNA-binding proteins may prove to be a moregeneral strategy for the posttranscriptional control of miRNAbiogenesis. Previous reports have demonstrated that nucleoli containmachinery responsible for modifying small nucleolar RNAs, for examplethrough RNA methylation or 3′ uridylation. Whether these nucleolarmechanisms play a role in pri-miRNA regulation remains to be determined.Boisvert et al., 2007; Matera et al., 2007. It is possible thatadditional new factors may be involved in sequestering and possiblydegrading and/or modifying pri-let-7 miRNAs in the nucleoli in aLin28B-dependent manner. The identification of such factors could revealnew potential therapeutic targets aimed at restoring let-7 expression inLin28B-expressing cancers.

The present proof-of-concept studies with human breast and ovariancancer cell lines demonstrate that inhibition of Zccch11 may represent apossible new therapeutic target for cancer. Indeed, knockdown of Zcchc11effectively inhibits the tumorigenic capacity and metastatic potentialof human breast and ovarian cancer cells and xenografts. Importantlyhowever, the present work also predicts that therapeutic potential ofZcchc11 inhibition is particularly relevant in Lin28A-expressingcancers. Although Lin28A expression is relatively uncommon in severalhuman cancer cell lines, the analysis of primary human colon and breastcancer indicate that upregulation of Lin28A or Lin28B occurs in a largeproportion of tumors with approximately equally frequency for eachprotein. Furthermore, expression of Lin28A or Lin28B seems todistinguish different classes of breast cancers (FIG. 20). Therefore,the identification of small molecule inhibitors of Zcchc11 may lead tothe development of novel chemotherapies for Lin28A-expressing cancers.

Recent work examining the role of miRNAs in development and cancer hasrevealed extensive post-transcriptional control at various levels ofmiRNA biogenesis. Siomi & Siomi, (2010). Lin28A and Lin28B have emergedas important posttranscriptional regulators of let-7 expression in stemcells, development, metabolism, and disease. Viswanathan & Daley, 2010.In the case of Lin28A, this regulation involves the recruitment of aTUTase Zcchc11 to catalyze the 3′ terminal uridylation of pre-let-7RNAs. Several studies have identified extensive non-templated nucleotideaddition to the 3′ ends of mature and precursor miRNAs. Ameres et al.,2010; Berezikov et al., 21 Genome Res. 203 (2011); Burroughs et al., 20Genome Res. 1398 (2010); Chiang et al., 24 Genes Devel. 992 (2010); Heoet al., 2008; Jones et al., 2009; Katoh et al., 23 Genes DEvel. 433(2009); Lehrbach et al., 2009; Newman et al., 2011. The presentembodiments provide the first extensive mechanistic analysis of one ofthese enzymes, Zcchc11.

More specifically, specific TUTase domains that are required formediating efficient Lin28-endhanced uridylation of pre-let-7 in vitrohave been uncovered. Of the four zinc fingers encoded in Zcchc11, theunique C2H2 zinc finger at the N-terminus of the protein mediates theinteraction with Lin28 as point mutations in conserved Cysteine residuesof this zinc finger abolish Lin28-enhanced uridylation activity. TheTRF4 domain at the N-terminus of Zcchc11, while incapable of supportinguridylation activity on its own, is nonetheless required for activity invitro. This essential role may explain its significant degree ofconservation across taxa. Furthermore, the CCHC zinc fingers, whichdefine a class of at least thirteen mammalian proteins, aredifferentially required for uridylation activity in vitro. Specifically,the C-terminal-most CCHC zinc finger is dispensable for in vitroactivity, while the zinc finger just C-terminal to the active site isrequired for any detectable activity. Further, there are regionsdispensable for Lin28-enhanced uridylation at the N- and C-termini (FIG.14C). Both of these domains are of unknown function but remain conservedin other organisms.

Because Zcchc11 has been implicated in several other biologicalpathways, the domains identified as dispensable may be required forother processes. Indeed, a recent study has identified the N-terminalportion of Zcchc11 similar to the N-PAP construct studied above (FIG.14A) to be sufficient to alter the cell cycle of cultured human cancercells. Blahna et al., (2011). The present invention unveils criticaldomains and residues that are required for Lin28-dependent Zcchc11activity. Though still controversial, the Lin28-mediated control oflet-7 expression in C. elegans has also been reported to involvepre-let-7 uridylation. Lehrbach et al., 2009; Van Wynsberghe et al.,2011. Notably, the proposed Zcchc11 ortholog, PUP-2, lacks the C2H2domain that mediates the functional interaction between Lin28 andZcchc11 as found herein. Lehrbach et al., 2009. Therefore it remainsunclear if and how worm Lin28 recruits PUP-2 to repress let-7expression.

The N-terminal C2H2 zinc finger of Zcchc11 synergizes with Lin28 to bindpre-let-7g. The requirements of pre-let-7 that mediate Lin28-enhancedactivity show that the preE of let-7g to be sufficient to direct thisactivity. Although Zcchc11 recognizes and uridylates the 3′-end ofpre-let-7 family members and other miRNAs, this occurs through amechanism that is independent of sequence information proximal to thesite of uridylation. Instead, Lin28 bound to an intact preE sequence issufficient to direct robust uridylation of the pre-miRNA. Although thepreE used herein contains the Lin28-binding motif of GGAG, this sequencewas previously shown to be insufficient in directing uridylationactivity towards pre-let-7, as gain-of-function experiments indicatedthe positioning of the motif relative to the Dicer cleavage site wasalso essential. Heo et al., 2009. The chimeric pre-21S7L, however, hasthe GGAG motif positioned not in the preferred site ending fournucloetides (4 nt) before the Dicer cleavage site, but only twonucleotides away from this point, suggesting there are other sequence orstructural determinants directing Zcchc11-mediated uridylation againstpre-let-7 miRNAs. The role of other protein factors giving furtherspecificity can be ruled out because the reaction could be reconstitutedfrom recombinant proteins produced in bacteria, but what defines thislevel of specificity remains unknown.

Structural studies have uncovered the degree to which the let-7 preE isaltered by bound Lin28, revealing a partial unwinding of the duplexregion near the site of Dicer cleavage. Nam et al., 2011. Although it isunknown how far this melting proceeds into the stem of pre-let-7, thisstructural change could alter the RNA so that it is a preferredsubstrate of Zcchc11. Indeed, a recent structural study showed that theCCHC zinc fingers of Lin28 preferentially bind the single strandedheptad sequence of AGGAGAU (SEQ ID NO:3) in the stem of pre-let-7,providing evidence of sequence-specific RNA binding by zincfinger-containing proteins. Loughlin et al., Nat. Str. Mol. Biol. 2011.Alternatively, pre-let-7-bound Lin28 undergoes a conformational changeand this may provide a suitable protein-protein interaction surfacebetween the Lin28-let-7 complex and Zcchc11. Nam et al., 2011. Moredetailed RNA mutagenesis and/or structural studies examining theinterplay between pre-let-7 and these two RNA binding proteins mayprovide additional insight into precisely how Lin28 functionallyenhances C2H2 binding to the let-7 preE. The uridylation and adenylationof mature miRNAs by Zcchc11 has also been reported. Jones et al., 2009;Wyman et al., 2011. Because Zcchc11 exhibits similar basal activitytowards unrelated pre-miRNAs (FIG. 17B) there may be othersequence-specific recognition factors that guide Zcchc11 activitytowards other RNA substrates including mature miRNAs.

The findings in the present mutational analysis led to the investigationof other putative TUTases, and to the identification of Zcchc6 as aregulator of let-7 expression. One study investigating the potentialredundancy between Zcchc11 and Zcchc6 found that only Zcchc11 wascapable of binding stem-loop containing histone mRNAs, while Zcchc6appeared to lack this capacity. Schmidt et al., 2011. In the case ofLin28 and let-7, however, Zcchc6 functioned identically to Zcchc11 invitro as its enzymatic activity against a synthetic let-7 precursor wasenhanced by either Lin28A or Lin28B, as was previously shown forZcchc11. Heo et al., 2009. Furthermore, Zcchc6 is crucial in efficientlyrepressing mature let-7 miRNAs in embryonic cells. Although the doubleknockdown of Zcchc11 and Zcchc6 led to more dramatic let-7 derepressionthan the loss of either TUTase alone, it still did not reach the levelsobserved upon Lin28A knockdown. This could be explained by incompleteknockdown of both TUTases or the activity of other as-yet unidentifiedlet-7 repressive factors.

The identification of a second TUTase regulating let-7 turnover mayprovide valuable insight into the control of let-7 expression in cancerand embryonic stem cell biology. The expression pattern and localizationof Zcchc6 are unknown but it is possible that the relative expressionlevels of these two redundant TUTases will determine the relativecontribution of Zcchc6 and Zcchc11 in the Lin28A-mediated control oflet-7 expression. In this regard, the present invention shows thatZcchc11 inhibition in Lin28A-driven cancers can block tumor growth invitro and in vivo, and Zcchc6 may have relevance in this context.Whereas Lin28 proteins might present particular challenges aschemotherapeutic targets due to their non-enzymatic activity, Zcchc11(and potentially Zcchc6) poses an intriguing possibility as a drugtarget because of its defined active site and the available structuraldata regarding non-canonical poly(A) polymerases. Stagno et al., 399 J.Mol. Biol. 464 (2010). Moreover, the ability to reconstitute thisregulatory pathway with recombinant proteins, as shown herein, providesan opportunity to perform in vitro screening to identify small moleculeinhibitors of TUTase activity as potential new chemotherapeutic agents.These possibilities allow expansion upon the novel centrality ofuridylation in stem cell maintenance and tumor development.

The present invention also provides for modulation of TUTase activities,particularly as these enzymes related to oncogenic pathways. Forexample, the present embodiments provide for a detailed biochemicalanalysis including the isolation and characterization TUTase complexes.These experiments provide a detailed understanding of the mechanism ofthis newly identified oncogenic pathway and is highly relevant to avariety of different Lin28A-expressing human malignancies includingBreast, Colon, Ovarian, and Germ cell tumors. The methods describedherein are applicable to the screening and characterization ofLin28-TUTase expression, for example, in a large cohort of breasttumors. This information consolidates Lin28 and the TUTase as importantdiagnostics as well as cancer therapeutic targets.

Emerging data, such as that shown herein, indicate that RNAoligouridylation represents an important aspect of regulated geneexpression. The global extent of oligo(U) addition throughout thetranscriptome remains to be determined, and the widespread relevance ofthis RNA modification in the posttranscriptional control of geneexpression is unknown. The human and mouse genomes contain seven knownor predicted TUTases. Individual TUTases may selectively modify a subsetof RNAs. Recent data have demonstrated a role for certain TUTase familymembers in: (a) the control of histone gene expression during the cellcycle (TUTase 1 and 3), (b) the recycling of small nuclear U6 RNA (U6TUTase), (c) the Lin28-mediated regulation of let-7 microRNA biogenesisin embryonic stem (ES) cells (TUTase 4), (d) gene silencing by miRNA andsiRNA that apparently involves uridylation and also for the control ofmature microRNA stability/function, (e) mRNA bulk turnover managed bythe fission yeast Cid1 (a putative ortholog of TUTase 7 in mammals), and(f) maintenance of genome stability requiring the uridylation activityof Cde-1/Cid1 gene in C. elegans. When taken together, these reportssuggest that certain TUTases may have widespread roles in controllinggene expression and transcript stability. The complete repertoire of RNAunder the regulation of each of the known and predicted TUTases remainsunknown, however. As shown herein regarding Zcchc11 and Zcchc6, theseenzymes display a strong sequence preference for uridylating a subset ofmature miRNAs that includes the tumor suppressor let-7 family as well asthree other families of miRNAs that have been implicated with varioushuman malignancies. Considering these together with recently publishedhigh throughput sequence analyses of miRNAs that identify extensive 3′terminal uridylation of certain miRNAs supports the conclusion thatselective uridylation of mature miRNAs with tumor suppressor function,for example by Zccch11 and Zcchc6, represents an important new oncogenicpathway. The present invention will identify other miRNAs that aredirectly modified by these paralogous TUTases, explore the functionalconsequences of Zcchc11/Zcchc6-catalyzed 3′ terminal uridylation ofthese miRNAs, and illuminate the importance of this pathway in humancancer. Cell based assays as well as xenograft experiments with bothTUTase loss- and gain-of-function will reveal the impact of these newlyidentified enzymes to human cancer cell biology and will help uncovernovel oncogenic pathways the extent of which will be investigated inprimary human tumors. Finally, key downstream miRNAs and their targetgenes will be identified through functional studies to identify thosethat lead to oncogenic cellular transformation.

Screening can include RNAi-mediated loss of function experiments todeplete TUTases, such as Zcchc11 or Zcchc6, in Lin28A-expressing cancercells and monitor let-7 expression by qRT-PCR and Northern Blot. Thecellular consequences of TUTase knockdown including cell proliferationand colony formation assays can also be examined. A panel oflentiviruses that express shRNAs targeting Lin28A, Zcchc11, and Zcchc6has been generated. These reagents provide a valuable set of tools toeffectively deplete the expression of the relevant proteins in humancancer cells. Thus, these shRNAs can be deployed to examine themolecular and cellular effects of TUTase knockdown in human cancercells. Importantly, these shRNA constructs are designed to enable theknockdown of both Zcchc11 and Zcchc6 TUTases simultaneously to addressany possible redundancy. For this, the shZcchc11 construct includes apuromycin resistance gene and the shZcchc6 construct includes ahygromycin resistance gene. This provides for double antibioticresistance in transduced cells for knockdown of both Zcchc11 and Zcchc6.The expression pattern of Zcchc11 and Zcchc6 is largely unknown, butrelative expression levels of these two redundant TUTases may determinethe relative contribution of Zcchc6 and Zcchc11 in the Lin28A-mediatedcontrol of let-7 expression. In this regard, as shown herein, Zcchc11inhibition in Lin28A-driven cancers can block tumor growth. It istherefore important to explore the relevance of Zcchc6 and other TUTasesin this context.

In accord with the work presented herein related to Zcchc11, tounderstand which domains of TUTases are required for uridylationactivity, we will generate a series of mutant cDNAs and test the abilityof the resulting Flag-immunopurified (Flag IP) proteins to uridylatesynthetic pre-let-7 miRNA in vitro. For example, TUTase mutants will begenerated lacking N- and C-terminal domains or harboring point mutationsin conserved residues. As described herein, Zcchc11 exhibits a low-levelof uridylation against pre-let-7 and this activity is strongly enhancedby the addition of recombinant or immunopurified Lin28. As determined bythe incorporation of radiolabeled UTP, the activity of wild type TUTasesis compared with each of the series of mutant TUTases. These studiesprovide insight into the basic mechanism underlying the catalytic natureof these TUTases, and identify the minimal domains are preserved invarious TUTases.

Additionally, the present embodiments provide for the determination ofRNA structural and sequence determinants of TUTase regulation of miRNAs,such as pre-let-7, using a variety of approaches including EMSA, RNasefootprinting, and TUTase activity assays with a panel of different RNAsubstrates. For example, evidence that Zcchc11 requires its N-terminalC2H2 zinc finger to mediate Lin28-enhanced uridylation suggested thatthis region is important for physically interacting with either Lin28 orrecognizing a Lin28-bound form of pre-let-7. This model can be appliedto other miRNA models, using electrophoretic mobility shift assays(EMSA) with recombinant 6×-His Lin28 and a fragment of the TUTasecontaining the site required for synergism, for example, a C2H2 zincfinger (r.C2H2). The relevant fragment is expressed and purified frombacteria as a recombinant protein. As previously shown, incubatingr.Lin28 with 5′ end-labeled pre-let-7 leads to the formation of a stablecomplex that can be resolved on a native polyacrylamide gel.

EMSA can be used to measure the relative ability of miRNA-bindingproteins and TUTases to bind the respective miRNA, and to explore thepossible formation of a ternary ribonucleoprotein complex. Cooperativebinding between the miRNA-binding protein (e.g., Lin28) and the TUTasefragment (e.g., C2H2 fragment) can also be explored by performing EMSAexperiments with combinations of both proteins at subsaturatingconcentrations; and further using fragments of the miRNA (e.g.,fragments of pre-let-7 RNA). In the model provided by the instantinvention, the terminal loop region of the miRNA that is removed byDicer, more recently termed the pre-element or preE, is useful.

Additionally, RNase footprinting experiments can investigate changes inRNA structure upon binding by the miRNA-binding protein and criticalTUTase fragment (e.g., Lin28 and C2H2). This provides insight into thedynamic changes in protein-RNA and/or protein-protein interactions thatmight help explain the functional interaction between Lin28 and Zcchc11leading to the Lin28-enhanced TUTase activity. 5′end-labelled pre-let-7gcan be analyzed by RNase digestion. Recombinant miRNA-binding proteinsand recombinant TUTase proteins are titrated into these in vitroreactions and subsequently perform the RNase digestions. DifferentRNAses with diverse specificities allows detection of specific cleavagesites that are protected from digestion by the dual proteins, thusindicative of protein binding. Investigating which cis-acting RNAelements support uridylation enhanced by the miRNA-binding proteinreveals the role of pre-miRNA substrates in TUTase-mediated uridylation.For example, Lin28 binding to pre-let-7 requires specific sequence andstructural information in both the RNA and the protein.

TUTases will be purified from cells and interacting proteins identifiedto define the composition of TUTase-containing ribonucleoproteincomplexes. Biochemical reconstitution assays as well as RNAi-mediatedloss-of-function experiments will be used to explore the role of TUTasecomplex components in the control of miRNA expression and effects ongrowth and tumorigenicity of miRNA-derepression-associated cancer celllines. For example, Zcchc11 is present in ˜600 KDa protein complex,indicating that the ˜180 KDa Zcchc11 protein is a component of amulti-subunit protein complex. In order to gain insight into themechanism by which TUTases regulate miRNA processing it will benecessary to identify TUTase-interacting proteins. TUTase-asssoicatedproteins are identified, to experimentally confirm these interactions,and to assigned to particular complexes. The identification ofTUTase-binding partners will enable the biochemically definition ofcytoplasmic TUTase complexes and will help uncover the mechanism ofposttranscriptional regulation of miRNA-processing in human cancercells.

Biochemial definition of the composition of rTUTase-containing complexescan be done as shown herein for Zcchc11. Sable cell lines expressingFlag-TUTases are derived, and clones expressing high levels ofFlag-tagged protein selected and expanded for large-scale purifications.This approach is an extensively utilized methodology in the lab forprotein complex purifications. Because TUTases such as Zcchc11 localizeprimarily to the cell cytoplasm, it may be necessary to isolate andcharacterize cytoplasmic TUTase-containing complexes. This oftenrequires >100 mg of protein extract to isolate and identify theTUTase-interacting proteins, which is achieved by growing 100×15 cmplates of Flag-TUTase cells and preparing S100 and nuclear extract. Theanticipated protein extract yield is around 200-300 mg S100 extract,from which Flag-TUTase associated proteins are immunoprecipitated. Afterextensive washes, the Flag-affinity purified material iseluted from thebeads using Flag peptide. The protein eluates are typically analyzed byFlag western blot to confirm the purification.

Next, a sample of affinity eluates is resolved by 4%-15% SDS-PAGE andanalyzed by silver staining. The silver staining of the affinity eluatereveals coeluting polypeptides. In parallel, immunoprecipitation ofnaïve cells serves as a control. Comparison of this ‘mock’ withFlag-TUTase immunoprecipitation will identify bands specificallyco-eluting with the TUTases and distinguish them from bands representingany contaminating polypeptides. Precipitation the affinity eluate withtrichloroacetic acid (TCA) and resolution of the sample by SDS-PAGE isfollowed by colloidal blue staining. Individual polypeptides are excisedfrom the gel and subjected to mass spectrometric sequencing performed.The polypeptides that co-purify with the TUTases are sequenced, and thisinformation gives insight into the mechanism of TUTase function. It islikely that several of these peptides correspond to gene products thatfunction in various aspects of RNA metabolism including DEAD box or DEAHbox RNA helicases, RNA recognition motifs (RRM), exo- andendo-nucleases, heterogeneous nuclear ribonucleoproteins (HNRPs), orother protein family members involved in RNA-processing and/orstability. In addition, this unbiased purification strategy may yieldnovel proteins without obvious motifs whose function to date hasremained obscure.

Considerable care is taken to determine whether proteins that areidentified in the these studies are bona fide components of the TUTasecomplexes. Several approaches can determine whether a specific proteinis tightly associated with a TUTase. For example, antisera against newproteins confirms wheterh they are present in the immunoprecipitants ofFlag antibodies. Also, whether the new proteins co-elute with the TUTaseon a gel filtration column is shown by examining the fractions by silverstain and western blot. Further, one can assay for reciprocalco-immunoprecipitation of the new proteins with the TUTase.Additionally, individual cDNAs of some of the new proteins are subclonedto a vector for overexpression of Flag-tagged fusion protein inmammalian cells, and used for the generation of stable cell lines. Afterscreening for expressing clones, cells are expanded for performlarge-scale affinity purifications and analyses as described herein.Collectively, these experiments enable establishment of the polypeptidecomposition of the TUTase complexes and provide the foundation for adetailed functional analysis.

Some of the identified components of the TUTase complex will likelyaffect miRNA-binding protein/TUTase activity in vitro. The in vitroTUTase activity assay and biochemical reconstitution experiments withpurified components to identify factors that are required formiRNA-binding protein-enhanced pre-let-7 uridylation have beenestablished herein. Performing in vitro TUTase assays using cellextracts prepared from control cells and from cells in which complexcomponents have been depleted by RNAi will be a complementary strategyto identify a subset of factors that are specifically required for theaction of the TUTase complex. Further assays described herein explorethe role of TUTase complex components in the control of let-7 expressionand effects on growth and tumorigenicity of Lin28A-expressing cancercell lines. For these experiments, miRNA binding pretein-expressingcancer cells including T47D (Breast ductal epithelial tumor), and IGROV1(Ovarian carcinoma) cancer cells are infected with lentivirusesexpressing shRNAs to knockdown expression of the relevant genes (e.g.,the miRNA-binding protein or the TUTase complex components). For boththese well-characterized adherent cancer cell lines, data shows thatLin28A or Zcchc11 depletion leads to elevated let-7 expression andinhibits the colony formation, metastatic potential, and tumorigeniccapacity in knockdown cells.

For genes that modulate miRNA-binding protein-TUTase activity,corresponding changes in let-7 miRNA levels are expected when thesegenes are depleted in cells. Total RNA is extracted from knockdown cellsand q.RT-PCR performed (using the TaqMan® system from AppliedBiosystems) to measure relative miRNA levels. These assays are performedin parallel for several miRNAs, at least some of which serve ascontrols. The efficacy of target gene knockdown is confirmed bymonitoring the expression of miRNA-binding protein, TUTase and otherTUTase complex components, by performing western blots (or by q.RT-PCRif antibodies are unavailable) on cells treated with the individualshRNAs and controls. To reduce any possible off-target effects we willuse multiple (at least two) different shRNA or siRNA to knockdown theexpression of each target gene. Cell Proliferation, Colony Formation,Invasion, Xenograft Assays on control and knockdown cells (i.e., Lin28Aor the TUTase complex components) are performed as described herein.

The present data indicate that Zcchc11 and Zcchc6 not only function withLin28A to uridylate let-7 precursor RNAs but also selectively uridylatea subset of mature miRNAs in a Lin28-independent manner. The centralhypothesis is that Zcchc11/Zcchc6 regulate miRNAs through sequencespecific uridylation of mature miRNAs to regulate their stability and/orfunction. Thus, TUTase activity assays are performed with a panel ofsynthetic RNAs to determine the RNA sequence requirements for Zcchc11-and Zcchc6-mediated uridylation of mature miRNAs. Results from theTUTase activity assays performed using different RNA substratesuncovered a strong preference of the TUTases Zcchc11 and Zcchc6 forcertain mature miRNA sequences. In particular, the mature let-7 miRNAwas a preferred substrate for both Zcchc11 (FIGS. 21A-B) and Zcchc6.

To begin to address the RNA sequence determinants for this substratepreference the primary sequence of let-7 family members was examined.This revealed several regions of conservation, including (as expected)the 5′ seed sequence (nt 2-8) as well as other sequence motifs conservedbetween different let-7 family members and between mouse and human let-7miRNAs. To determine if any of these sequences were necessary for theRNA preference displayed by Zcchc11 in previous experiments, mutant RNAoligos were designed and tested in vitro. Mutations in the seed andcentral sequence slightly reduced the substrate preference of Zcchc11(FIGS. 21A-B). Mutations in the central and 3′ regions, as well as anRNA oligo that had all three domains mutated, dramatically reducedsubstrate preference.

Because other RNA-binding proteins with CCHC Zinc fingers preferentiallybind GXXG motifs, Zcchc11 might use a similar G-rich motif to recognizesequence information in let-7 guide strands. Interestingly, eliminatingall guanine residues in a let-7 guide oligo also severely compromisedits ability to serve as a strong Zcchc11 substrate. To confirm thesufficiency of these sequence motifs for substrate preference, an RNAoligo that contained nine nucleotides from the center of let-7 guidewere inserted into an unrelated RNA, the Luciferase GL2 siRNA sequence.The activity of Zcchc11 towards this chimeric RNA oligo resembled let-7guide, and was a drastically better substrate compared to the parentalGL2 RNA oligo. Taken together, these results indicate that Zcchc11uridylates single-stranded RNA in a sequence-specific manner and thatthis sequence is both necessary and sufficient to confer a strongsubstrate preference.

A bioinformatic search of all (>1,000) annotated miRNAs in the mouse andhuman genomes identifies miRNAs that may be similarly recognized byZcchc11. Because two miRNAs, let-7g and let-7i, contained the perfectsequence GUAGUUUGU (SEQ ID NO:4) was sufficient to confer Zchcc11substrate specificity, the search was expanded by selecting miRNAs thatcontained both GUAG and UUGU sequences. A list of all miRNAs with thesesequences common to both the human and mouse was generated. Aside fromincluding eight of nine unique let-7 family members, this new listincludes seven additional miRNAs that comprise three distinct miRNAfamilies. Several of these miRNAs are known to directly regulate Hoxgene expression, including the miR-10, miR-99/100, and the miR-196families. miR-10a, miR-10b, miR-196a, and miR-196b are encoded withinHox gene clusters, and miR-100 and miR-99a, miR-99b are not. Along withnearly the entire let-7 family, the sequence-specific preference ofZcchc11 and Zcchc6 for these developmentally essential miRNAs may pose acrucial and evolutionarily ancient mode of regulating miRNAs to controldevelopment. Importantly, the altered expression of several of thesemiRNAs is linked with various cancers. Synthetic miR-10a resembles let-7guide in uridylation assays, and mutations in its conserved motifabolishes this preference (FIGS. 21A-B). Similar experiments with avariety of miRNA substrates provides a catalog of the exact sequencepreferences of these TUTases, and correlates the data from these invitro experiments with data generated from the RNA cloning and deepsequencing.

Another step generates small RNA cDNA libraries from control and TUTaseknockdown samples and analyzes the changes in miRNA expression and 3′terminal uridylation by high throughput cDNA sequencing technology.Recent deep sequencing analyses have revealed extensive non-templated(Adenylation or Uridylation) nucleotide addition at the 3′-end ofcertain mature miRNAs. Though the functional relevance of thesemodifications and the identity of the RNA terminal transferasesmediating these events remain unknown the extent of terminal uridylationis quite extensive for certain miRNAs and seems dynamically regulated indifferent tissues and mouse developmental stages. A large data set thatwas recently published from the Bartel lab includes 60 million small RNAsequences cloned from mouse brain, ovary, testes, embryonic stem cells,three embryonic stages, and whole newborns. This data set providesevidence for extensive uridylation of certain miRNAs, including thosemiRNAs that are preferred substrates for Zcchc11 and Zcchc6 in vitro,whereas other miRNAs such as miR-21 seem to be much less modified (FIG.22). miR-122, a miRNA that is adenylated by a poly(A) polymerase GLD-1is included in our analysis since it has been reported that 3′adenylation stabilizes miR-122 levels. An established protocol for RNAcloning and sequencing small RNAs from control and TUTase-depleted cellsis used. The data generated helps guide the choice of cell lines, i.e.,those cancer cell lines with elevated Zcchc11 and/or Zcchc6 expression.A useful cell line is MCF7 breast cancer cells, which express Zcchc11but not Lin28A or Lin28B. The basic procedure for small RNAs cloning isestablished. High throughput screening (using either the Illumina orSOLiD/Applied Biosystems platforms) is performed. This experimentaldesign includes conditions in which each TUTase is knocked downindividually as well as combinatorialy to address possible functionalredundancy.

Changes miRNA expression is measures, and reporter gene assays monitormiRNA function to identify the consequences of 3′ terminal uridylationof mature miRNAs. The experiments identify the extent ofTUTase-catalyzed miRNA uridylation in human cancer cell lines. Thesedata provide the foundation for experiments designed to address thefunctional consequences of miRNA terminal uridylation. Possibilities formiRNA regulation include (a) 3′ terminal uridylation regulates miRNAlevels, or (b) 3′ terminal uridylation regulates miRNA function withoutaltering expression of the miRNA. Although the study of these regulatorypathways is still in its infancy, there is precedence supporting both ofthese possibilities. These models are tested using q.RT-PCR and Northernblot to monitor possible changes in miRNA expression in TUTase-depletedcells. RNA uridylation targets miRNAs for rapid decay leads to increasedmiRNA levels in TUTase-depleted cells, whereas the levels of uridylatedmiRNAs should decrease. Taqman q.RT-PCR assays specifically detecturidylated versions of the mature miRNAs. This serves as a useful toolproviding a facile and quantitative means to monitor changes in miRNAuridylation in response to TUTase modulation, and complements thecommercially available assays that specifically detect thenon-uridylated miRNAs. If miRNA expression is not affected byTUTaseknockdown, then miRNA function is tested. miRNA-responsive reportersmeasure miRNA-mediated posttranscriptional gene silencing. Control andTUTase knockdown cells transfected with Luciferase reporters bearingrelevant miRNA-target sequences in the 3′UTR, and the extent of themiRNA-mediated repression is measured by comparing relative Luciferaselevels.

If the TUTase(s) impact miRNA function without altering miRNA expressionlevels, then the molecular basis for this regulatory mechanism isexplored. miRNA uridylation may alter the miRNA association with theArgonaute proteins, this can be tested directly by affinity-purifyingArgonaute proteins from control- and TUTase-depleted cells and analyzingthe associated miRNA by q.RT-PCR and/or Northern blot. Similarexperiments to test directly the function of miRNA uridylation can beperformed by transfecting TUTase-depleted cells with control or 3′uridylated synthetic miRNAs and measuring Argonaute association as welltarget gene repression.

The present invention also provides for compositions and methods todetermine the oncogenic role of the TUTases. For example, Zcchc11 andZcchc6 selectively modulate the expression of a subset of miRNAsincluding the tumor suppressor let-7 family. This regulation likelyoccurs by both Lin28A-dependent as well as Lin28A-independentmechanisms. Thus, TUTases, such as Zcchc11 and Zcchc6, function asoncogenes and promote cellular transformation. The relevance and scopeof this regulatory pathway in cancer biology is analyzed using Lin28A,Lin28B, Zcchc11, and Zcchc6 expression in cancer cell lines, primaryhuman tumors, and corresponding normal tissues using q.RT-PCR, Westernblot, and immunohistochemistry. miRNA expression is measured in the samesamples by q.RT-PCR and in situ hybridization. Lin28A and Lin28Bexpression was measured in small cohort of human colon and breasttissues, revealing that Lin28A or Lin28B were upregulated while let-7awas downregulated in colon adenocarcinomas relative to normal colontissues. In addition, Lin28A was significantly upregulated inHER2-overexpressing breast tumors, while Lin28B was significantlyupregulated in triple-negative (ER-,PR-,HER2-) breast tumors. Thebroader relevance of these findings in breast cancer is extended bymeasuring Lin28A, Lin28B, and TUTases (such as Zcchc11 and Zcch6) in amuch larger cohort of breast tumor samples.

Tumor tissue microarrays are obtained from, for example, the HumanGenetics Sample Bank at Ohio State University. These tumor array samplesfrom over 800 patients are readily available as well as correspondingRNA samples from the tumor and adjacent normal breast tissues. Thisallows measurement of miRNA-binding protein and TUTase (e.g., Lin28A,Lin28B, Zcchc11, and Zcchc6 protein) expression and miRNA levelsexpression in a large number of lobular and ductal breast carcinomas andnormal breast tissues. This allows determination of the prevalence ofthese genes expression in the two major histologic subtypes of breastcancer and to determine the association between expression of theseproteins with important histopathological parameters in breast cancerincluding ER, PR and HER2 status, grade, tumor size, stage, lymph nodestatus etc. For those samples with follow-up data, whether expressionlevels of miRNA-binding proteins and the TUTases are associated withoutcomes, such as relapse-free and overall survival, is determined. Inaddition to measuring expression of miRNA-binding protein and TUTaseparalogs, the expression levels of the most relevant miRNAs includinglet-7 si measured. Double-DIG labeled miRCURY LNA™ Detection probes(Exiqon) are used for specific miRNA detection by in situ hybridization.One skilled in the art can generate or buy custom rabbit polyclonalantibodies (Open Biosystems/Thermo Scientific), which can be thoroughlycharacterized. If developing antibodies for the target TUTase isunsuccessful, one can use q.RT-RCR to measure TUTase expression inbreast tissues.

In addition to breast cancers, prostate cancer are a highly relevanttumor. The Zcchc11 mRNA level is elevated in 18/30 (60%) of samplesanalyzed by q.RT-PCR, and Zcchc11 expression positively correlates withGleason score. Moreover, analysis of Zcchc6 Microarray expression datain ‘Oncomine’ revealed elevated (3.2 fold) expression in prostatecarcinoma (n=30) compared to normal prostate tissue (P value=3.13E-7) inthe data set from Tomlins et al., Nature Genetics 2007, thereby raisingthe possibility that these TUTases may function redundantly in prostatecancer. The proposed Zcchc11 and Zccch6 expression profiling guidesfunctional studies, as described herein.

RNAi-mediated loss-of function experiments are done in selected cancercell lines with elevated TUTase expression to characterize and monitorcell proliferation, colony formation, migration, and tumorigenicity. Apanel of assays described herein explore the role of the TUTases aspotential new oncogenes. For example, whether Zcchc11 and Zcchc6function as oncogenes in a Lin28A-independent context may be compared inbreast and/or prostate cancer cell lines with elevated Zcchc11 and/orZcchc6 expression that do not express Lin28A or Lin28B (for exampleMCF-7 breast adenocarcinoma cell line) and used for functional assaysdesigned to examine the role of the TUTases on cell growth andtumorigenicity. For these experiments Zcchc11, Zcchc6, or both Zcchc11and Zcchc6 (to test possible functional redundancy) are depleted usingshRNA, and the effects monitored as described herein.

TUTase gain-of function experiments are correlated with measured effectson cellular transformation. The ability of overexpression of theTUTases, such as Zcchc11 and Zccch6, to induce oncogenic cellulartransformation is tested directly, for example the effect of Zcchc11 orZcchc6 overexpression in MCF10A immortalized breast epithelial cells.Lin28A/B or Src overexpression is used as positive controls. Cells aretransduced with lentiviruses expressing the relevant cDNAs. MCF10A cellsmay become transformed within 48 hours after transduction, and TUTaseoverexpression may lead to a substantial change in cell morphology. Theeffects of Zcchc11/Zcchc6 overexpression is tested on the colonyformation ability of the transformed MCF10A cells. Xenograftexperiments, where control or transformed MCF10A cells are injected intoimmunodeficient (nu/nu) mice, test the propensity of the injected cellsto form of tumors. Overall, these experiments are designed test thehypothesis that elevated TUTase expression leads to cellulartransformation.

Further, the mechanism for oncogenesis is explored by examining theinvolvement of key downstream miRNAs and their respective target mRNAs.The experiments proposed herein may establish the TUTases (Zcchc11 andZcchc6) as new oncogenes in breast and/or prostate cancer. It may beimportant to investigate the mechanism for this oncogenic function andto dissect the gene regulatory pathways that are dysregulated by TUTaseoverexpression in cancer. Genetic rescue experiments are performed,where TUTase levels are manipulated by shRNA-mediated knockdown and,simultaneously, either inhibit the function of individual miRNAs usingantisense oligonucleotides to suppress miRNA function (miRCURY LNAantagomiRs from Exiqon) or overexpress individual miRNAs using syntheticmiRNA mimics or overexpression plasmids, and monitor the effects oncancer cell growth. Microarrays (e.g., Affymetrix) can be used tomeasure changes in gene expression in response to manipulation of TUTaselevels. Whether these TUTases regulate miRNA expression or functionremains to be determined, however in either case the levels of thetargets of these miRNAs will be altered. mRNAs with reciprocalexpression patterns in the TUTase knockdown and overexpression samplesmay be considered candidate target genes.

To better distinguish between primary targets and secondarytranscriptional effects, Argonaute protein is immunopreciptitated fromcontrol and TUTase manipulated samples and the co-purified RNA subjectedto RNA-Seq analysis. RNA is isolated from Argonaute immunoprecipitatesand multiplexed libraries are generated and sequenced. Reporter geneassays confirm the direct miRNA regulation of selected candidate mRNAs.Luciferase reporter constructs are generated in which the 3′UTR of thecandidate gene is subcloned downstream of a luciferase open readingframe. Transient co-transfection of these reporter constructs with themiRNA mimic or anitisense inhibitor, followed by analysis of luciferaseactivity will reveal whether these UTRs contain the necessary sequencesfor miRNA-mediated suppression. For reporters suppressed byco-transfection with the corresponding miRNA, the putative miRNA seedsequence(s) within the 3′UTR are mutagenized to confirm that the effectobserved is caused by a direct interaction. Finally, it is likely thatthis list of genes that are regulated coordinately with the TUTase willinclude known and novel genes with oncogenic function. This may beaddressed using shRNA to knockdown selected downstream genes andevaluate the impact of this suppression on TUTase-mediated oncogenictransformation.

In addition to cancers, the role of miRNA function in the heart has beenaddressed by conditionally inhibiting miRNA maturation in the murineheart, and has revealed that miRNAs play an essential role during itsdevelopment. miRNA expression profiling studies demonstrate thatexpression levels of specific miRNAs change in diseased human hearts,pointing to their involvement in cardiomyopathies. Furthermore, studieson specific miRNAs in animal models have identified distinct roles formiRNAs both during heart development and under pathological conditions,including the regulation of key factors important for cardiogenesis, thehypertrophic growth response, and cardiac conductance. Further, miRNAsappear to regulate the nervous system. Neural miRNAs are involved atvarious stages of synaptic development, including dendritogenesis(involving miR-132, miR-134 and miR-124), synapse formation and synapsematuration (where miR-134 and miR-138 are thought to be involved). Somestudies find altered miRNA expression in schizophrenia.

An important aspect of the present invention provides for compositionsand methods for screening for drugs that restore miRNA expression incancer, that will likely lead to the development of novel strategieswith broad applicability for effective cancer treatment. Let-7 familymembers are down-regulated in many different tumors. The findingsreported herein imply that restoration of let-7 expression is a uniqueand powerful new strategy for cancer therapy. Lack of an appropriate,safe, and effective delivery methods for regulating let-7 expression,however, is currently a major drawback for implementing such a therapy.The present identification of a novel pathway that selectively inhibitsexpression of let-7 miRNAs provides an exciting opportunity for thedevelopment of novel chemotherapeutic strategies to restore let-7expression in cancer and supports the hypothesis that inhibition ofLin28A-Zcchc11 in cancer cells will elevate expression of the tumorsuppressor let-7 to its physiologic level, leading to reduced expressionof multiple let-7 target oncogenes and inhibition of cancer cellproliferation. Drugs that target the miRNA biogenesis pathway remainlargely uncharacterized, and no high throughput screening directedtowards let-7 miRNA biogenesis has been reported thus far. The presentinvention provides for the identification and characterization ofbioactive chemicals that relieve inhibition of the tumor suppressorlet-7 miRNA that is mediated by oncogenic Lin28A and its associatedTUTase Zcchc11. One way to identify such potential novel therapeutics isto develop and perform automated high-throughput screening (HTS) assays.The present invention provides for novel HTS approaches to identifydrugs that restore let-7 miRNA expression in Lin28A-expressing cancers.Two complementary assays for HTS are envisioned, with the goal ofmaximizing the chances of a successful outcome: a cell-based system tomonitor let-7 expression, and a biochemical assay to identify inhibitorsof the TUTase. Hit compounds are validated using a series of secondaryscreens; the effect of these compounds on cancer cells is examined. Thismethodology will allows for the translation of basic science discoveriesto the development of new and effective cancer treatments. Embodimentsprovide for the development of novel chemotherapeutic strategies torestore normal let-7 miRNA levels in tumors. “Hit” compounds from highthroughput screening will relieve the Lin28A-mediated block in let-7processing and identify the therapeutic potential of these compounds forhuman cancer, and thus support the development of novel drug treatmentsthat effectively target the undifferentiated cells within the tumor.

There are several advantages of targeting this pathway as a novelchemotherapeutic strategy: (a) Lin28A is normally expressed primarily inembryonic cells; therefore specific inhibitors of this pathway shouldoffer a good therapeutic window with limited side-effects of anypotential cancer treatments. (b) Lin28A is expressed self-renewing stemcells and so specific inhibitors of this pathway will likely target thepoorly differentiated and tumor initiating cells. (c) Lin28A selectivelyand posttranscriptionally inhibits expression of multiple members of thelet-7 family miRNAs; and so inhibition of the pathway should lead to thecoordinate upregulation of multiple let-7 family members. (d)Lin28A-mediated blockade of let-7 requires an enzymatically activeTUTase that should be relatively easy to target with small molecules—forexample, by using nucleotide analogs that are already used in the clinicfor effective treatment of other diseases. (e) Lin28A is a key componentof a feedback loop, so re-expression of let-7 will be self-reinforcing,leading to further elevation of let-7 expression.

A cell-based assay that monitors let-7 activity for HTS involvesgenerating and validating stable cell lines that express a let-7 sensorreporter gene. The cell-based screening utilizes a Luciferase reportergene, which was engineered to monitor let-7 expression in culturedcells. A Luciferase reporter mRNA harboring a let-7 target sequence inthe 3′ untranslated region (UTR) can be used to specifically monitorlet-7 activity. The psiCHECK-2 vector (Promega) allows generation ofreporter constructs containing the let-7 target sequences. This vectorhas been designed to enable the monitoring of changes in expression of agene of interest, e.g., a let-7 target, fused to a reporter gene.Renilla luciferase is used as a primary reporter gene that containsmultiple cloning sites in the 3′UTR to make the cloning of let-7 targetsequences possible. Renilla luciferase expression is driven by SV40promoter and uses a synthetic poly(A). The psiCHECK-2 vector alsopossesses a secondary firefly reporter expression cassette that consistsof an HSV-TK promoter, a synthetic firefly luciferase gene and an SV40late poly(A) signal. This cassette has been designed to serve as anintraplasmid normalization reporter. The Renilla luciferase signal thuscan be normalized to the firefly luciferase signal. This vector has beenwidely used as a sensor to test the ability of miRNA to target RNAsequences and to induce posttranscriptional repression of the targetedgenes. In order to generate a sensitive reporter assay, a construct inwhich three let-7 target sequences arranged in tandem was included inthe 3′UTR of the Renilla luciferase was generated. These sequencesshould induce destabilization of the Renilla luciferase transcripts togive a broad window between conditions where the let-7 is expressed oris inhibited by the Lin28 pathway. As a proof-of-principle, the reporterwas used to specifically monitor let-7 activity. The data indicate thatthis let-7 reporter is destabilized by let-7. (FIG. 23A).

Relative Luciferase activity was measured in transiently transfectedcells. For this, we initially chose Hela cells (that express high levelsof let-7 miRNA) as well as P19 embryonal carcinoma cells (in which let-7expression is blocked by Lin28A). As expected, there was a dramaticdestabilization of the let-7 sensor in Hela cells, whereas the samereporter was stable in P19 cells (FIG. 23B). Next, the human lungadenocarcinoma cell line, H1299 (that expresses high levels of Lin28B)was engineered to contain the psiCHECK-2 vector containing the let-7target sites. This stable H1299 cell line was transfected with plasmidfor the expression of a chimeric pri-miRNA in which the terminal loopsequence of pre-let-7 is replaced with that of miR-21 (a miRNA that isnot regulated by Lin28). This chimeric construct bypasses Lin28-mediatedregulation thus providing a convenient means to ectopically expresslet-7 in Lin28-expressing cell lines. As expected, expression of let-7in this H1299 stable cell line resulted in a robust (˜5-fold) repressionof the Luciferase reporter construct bearing let-7 complementary targetsites (FIG. 23C). Stable cells lines containing the let-7 sensor aregenerated and validated by measuring response to RNAi-mediatedLin28-TUTase depletion. RNAi approaches may be used to monitor theresponse of each reporter to Lin28A, and Zcchc11 inhibition.

The Dual Luciferase Reporter Assay System (Promega) will be used toprepare cell lysates and to measure sequentially the activity of fireflyand then Renilla luciferase. Measurements will be performed using aSynergy 2 luminometer with the Gen5 program (Biotek). The relative lightunits (RLU) collected for the Renilla luciferase will be normalized tothe firefly luciferase RLU. The stable cell line where Lin28A/Zcchc11inhibits let-7 expression shows relatively high levels of Renillaluciferase. Treatment of a chemical inhibitor that disruptsLin28A/Zcchc11 repression of let-7 will result in increased let-7 levelsand a decrease in relative Renilla luciferase levels. The datademonstrate that shRNA-mediated depletion of Lin28A or Zchcc11 in IGORV1cells leads to accumulation of let-7 expression (FIG. 24). Establishmentof a stable cell line is more convenient and preferred to transientlytransfected cells and effectively provides an unlimited source ofhomogeneous cells in order to achieve a good reproducibility in HTSapplications. H1299 (Lung) cancer cells were chosen because they expressendogenous Lin28B, but because Lin28A (but not Lin28B) employs theTUTase to selectively repress let-7 expression, one may useLin28A-expressing cancer cell lines exclusively, for example,well-characterized adherent cancer cell lines expressing Lin28A T47D(Breast ductal epithelial tumor), and IGROV1 (Ovarian carcinoma). Forboth these cell-types, Zcchc11 depletion inhibits the colony formation,metastatic potential, and tumorigenic capacity.

A transfection protocol is used wherein one plasmid corresponds to thereporter construct based on the psiCheck-2 vector, and the othercontains a puromycin resistance marker. Puromycin selection may beadvantageous over neomycin selection because of its more potent toxicityto naïve cells. Then, 48 hours after transfection, culture media isreplaced with media supplemented with 2.5 μg/mL puromycin and cells willbe cultured for 10 to 14 days, with daily replacement of the media toremove dead cells. Stable cell lines are generated from a monoclonalpopulation, necessitating cell colony isolation. Often, polyclonalpopulations of cell lines stably expressing transgenes tend to loseexpression of the transgenic cDNA. After transfection and cellselection, positive clones are screened using luciferase assay andknockdown experiments. The stable cell line with the most significantlevel of luciferase activity for both luciferases and the mostresponsive to let-7 levels upon either ectopic let-7 expression, orLin28A//Zcchc11 knockdown is selected. Several stable clones with thereporter construct containing the Renilla luciferase-3× let-7 have beenselected Significant activities are reproducibly recovered for eachluciferase and ˜5-fold change in Luciferase ratio is obtained withenforced let-7 expression (FIG. 23). Therefore, highly predictable andreproducible responses to let-7 expression and the 5-fold changes inrelative Luciferase represent a relatively broad window that provides aclear threshold between positive and negative responses, establishingthis strategy for HTS suitability.

A pilot screen using a library of several hundred bioactive compoundscan be performed in order to determine the suitability of thiscell-based reporter gene system in an automated setting. We willdetermine whether the stable cell line(s) generated represents avaluable tool for HTS to identify small molecules targeting the Lin28Apathway. The validated cell line are used for HTS of a library of up totwo thousand chemicals. A pilot screen can be carried out on the Biomollibrary from the Institute of Chemistry and Cell Biology (ICCB) atHarvard University. This “Known Bioactives” library (Biomol) set hasbeen validated using other screening assays. Ten thousand cells areplated (in 90 μl) per well of the 96-well assay plates, and controlreagents and library compounds will be added 24 hours later. Eachcompound is tested in duplicate at three different concentrations (0.1,1, 10 μM). After 48 hours, wells are washed and lysed (20 μl lysisbuffer) to allow for assessment of Renilla and Firefly Luciferase levelsusing the Dual-Luciferase Reporter Assay System (Promega) and a BiotekSynergy2 plate reader. The resulting data is expressed as log2(RLuc/Fluc). The Z′ is calculated for each plate:Z′=1−3*(σCTRL+σDMSO)/|μCTRL−μDMSO|.

Criteria for identifying compounds appear in the ‘hit’ list if theyeither have a strong activity at a particular concentration or aconsistent activity over at least two concentrations, can be devisedbecause few compounds can be expected to be active over the fulldilution range. Candidate hit compounds are validated using a series ofsecondary screens including q.RT-PCR analysis of let-7 expression,Western Blot analysis of Lin28A and Zcchc11 expression, and in vitroassays for TUTase activity. The most effective compound concentrationsis determined from dose curves; effects are monitored by Luciferaseactivity assays and by q.RT-PCR measurement of let-7 levels. This allowsdetermination of the stable cell line(s) generated represents as avaluable tool for HTS to identify small molecules targeting the Lin28 orTUTase (or both) pathway. Secondary screens may be performed toascertain compounds that specifically regulate Lin28A-TUTase complexactivity.

Putative hits from this primary screen can be evaluated throughsecondary screens that include dose curves, q.RT-PCR analysis of miRNAexpression, Western Blot analysis of Lin28A and Zcchc11 expression, andin vitro TUTase activity assays. These experiments allow for theelimination of false positives, and validation of compounds asregulators of the Lin28 pathway, and for exploration of their mechanismof action. Quantitative analysis of mature miRNA levels by q.RT-PCR maybe done as follows: Cells are treated with each compound selected as‘hits’ from the primary screen and levels of a let-7 miRNAs analyzed byq.RT-PCR. As shown herein, Lin28A or Zcchc11 knockdown led toaccumulation of let-7 in IGROV cells (FIG. 24), and T47D cells. Forcompounds that modulate Lin28-Zcchc11 expression or activity,corresponding changes in let-7 miRNA levels are expected. Total RNA isextracted from treated cells and q.RT-PCR will be performed using TaqManassays to measure let-7. These assays will be performed in parallel forseveral non-let-7 miRNAs such as miR-21 and miR-16 will serve ascontrols. Additionally, Western Blot Analysis of Lin28A and Zcchc11expression may be used. Hit compounds from the screen and validated toaffect specifically let-7 mature miRNA levels (by q.RT-PCR approachesdescribed above), likely target the Lin28 regulatory pathway in one oftwo ways; either by altering the expression of Lin28A, or Zcchc11 bysomehow influencing the activity of the Lin28-TUTase without directlyaffecting Lin28-TUTase expression levels. These alternativepossibilities are distinguished by secondary screening strategies.

This work allows exploration of the mechanism of action of hit compoundsand allows to elimination of compounds that may be acting indirectly.Therefore, the expression of Lin28A, and Zcchc11 is monitored by westernblots (and by q.RT-PCR) on cell treated with the individual compoundsand mock treated controls. Specific antibodies for the detection of eachof the endogenous proteins have been found (FIG. 24A). The correspondingexpression plasmids for transient overexpression of epitope taggedproteins are provided herein. These serve as useful tools in theinvestigation into the effect of compound treatment on Lin28A andZcchc11 expression. Utilizing the transgenes for ectopic proteinexpression, as well as analyzing endogenous Lin28A and Zcchc11 proteinand mRNA levels (by q.RT-PCR), enable one to distinguish betweenpotential transcriptional versus posttranscriptional regulation. Forexample, data reveal that Lin28 expression is repressed by let-7posttranscriptionally whereas other factors such as Myctranscriptionally activate Lin28A expression. Therefore, an analysis ofLin28A and Zcchc11 expression is a possible secondary screens.

In vitro TUTase assays can be employed also. Some of the hit compoundsmay influence Lin28A-TUTase activity independent of altered expressionlevels. An in vitro assay is used to identify chemicals thatspecifically inhibit miRNA biogenesis through targeting theLin28A-TUTase complex. For in vitro uridylation assays, syntheticpre-let-7 RNA is incubated with the affinity-purified proteins in thepresence of [α-³²P]-UTP. By adding individual chemicals identified inthe screen to biochemically reconstituted TUTase reactions with purifiedLin28A-Zcchc11 complex, or by performing in vitro TUTase assays usingcell extracts prepared from treated cells, a subset of chemicals isidentified that specifically inhibit the action of the Lin28A-Zcchc11complex and thereby promote let-7 biogenesis.

The optimized assay also provides for high throughput screening.Depending on the outcome of these pilot experiment, an additional˜400,000 compounds from a chemical diversity set can be screened. Thechemical screening can be performed using the services and chemicallibraries, such as that available at the Institute of Chemistry and CellBiology (ICCB)-Longwood, an investigator-initiated screening programthat assists academic researchers in carrying out high throughputscreening of chemical libraries. High throughput screening capabilitiesare also offered through the Harvard Stem Cell Institute. TheICCB-Longwood compound collection is continuously growing, and over400,000 compounds are currently available for screening. One can followthe recommended screening priority for compound libraries according tothe ICCB guidelines. The phase of this strategy is to screen the KnownBioactives libraries (6,671 compounds), followed by a selection of˜10,000 compounds from the Natural Product Extracts (48,688 compounds),a selection of ˜70,000 most recently plated compounds from theCommercial Libraries (223,041 compounds), followed by ˜50,000 recentlyplated compounds from the Commercial Libraries (223,041 compounds),˜38,000 remaining Natural Product Extracts (48,688 compounds), andfinally the remainder of the collection. Details of these libraries areavailable online. Depending on results from these screens, one maychoose to screen additional libraries available through The ChemicalBiology Platform of the Broad Institute. The platform team hashigh-throughput research capabilities in organic synthesis andsmall-molecule screening. Informatics and computational analysis teamsintegrate these capabilities. This library comprises over 500,000compounds and the Chemical Biology Platform participates in the NationalCancer Institute's Initiative for Chemical Genetics (ICG).

To establish, validate, and utilize a biochemical assay that monitorsTUTase activity for high throughput screening, one can design andoptimize in vitro TUTase activity assays using recombinant Lin28A andZcchc11 proteins and detection methods suitable for HTS, as describedherein. Because the activity of the TUTase is likely to be highly‘druggable’, the present invention also provides design, optimization,and tily of an in vitro HTS assay that monitors TUTase activity toidentify compounds that directly inhibit this enzyme. The reconstitutedthe in vitro uridylation assay is described herein. Briefly, syntheticpre-let-7 RNA was incubated with the affinity-purified Lin28 and Zcchc11proteins in the presence of [α-³²P]-UTP. To optimize this assay for HTS,non-radioactive detection methods are advantageous. The incorporation offluorescent nucleotides (UTP) or measuring levels of pyrophosphate (PPi)that is generated by nucleotide polymerization are alternatives (FIG.25). The detection of pyrophosphate (PPi) generated by nucleotidepolymerization is already commonly utilized for automated massivelyparallel DNA sequencing (deep-sequencing) technologies. Therefore weanticipate the adaptation of this pyrosequencing approach for monitoringTUTase activity should be feasible and enable high throughput screening.

An abundant supply of purified Lin28 and Zcchc11 (TUTase) proteins areused for the in vitro screening strategy. Highly purified recombinantproteins for these assays can be generated as taught herein. RecombinantLin28A is expressed in bacteria (His-Lin28A) and purified as described.Using this system, at least 2 mg of highly purified r.Lin28A can beobtained reproducibly from 1 liter of E. coli. For all TUTase activityassays so far described, the source of the TUTase has been Flag-Zcchc11affinity-purified from cultured human (HEK293) cells. For a HTSapproach, it may be necessary to establish conditions for the productionof recombinant Zcchc11 protein. The large size of the full-lengthZcchc11 protein (>180 kDa) is likely to be prohibitive for itsexpression and purification from E. coli, but the recombinant C2H2fragment described herein is advantageous. This truncated proteinretains full activity in the present pre-let-7 TUTase assays, andimportantly this minimal protein of <120 kDa, is substantially reducedfrom its original 184 kDa. By using this recombinant protein withrecombinant Lin28, the uridylation reaction can be reconstituted invitro, thereby defining the minimal components of Lin28-mediatedpre-let-7 uridylation and enabling the present innovative HTS approachto identify inhibitors of Zcchc11. The purified recombinant proteins arefunctionally validated by performing TUTase activity assays withradiolabeled pre-let-7 RNA. These proteins are used for the developmentof the non-radioactive assay. Once these recombinant proteins have beenvalidated, the assay is scaled to the 96- or 384-well format requiredfor HTS. Luciferase measurements can be performed using a Synergy 2luminometer with the Gen5 program (Biotek). The addition of individualchemicals to TUTase reactions that have been biochemically reconstitutedwith purified Lin28A-Zcchc11 complex should allow identification of asubset of chemicals that specifically inhibits the action of theLin28A-Zcchc11 complex and thereby promotes let-7 biogenesis.

A pilot screen is performed on the in vitro assay, using a chemicallibrary that contains a collection of nucleotide analogs. The catalyticactivity of the TUTase is inhibited by certain nucleotide analogs, andthat the identification of these compounds through HTS will lead totheir utility as a novel chemotherapeutic in Lin28A-expressing cancers.There are readily available libraries of such compounds; similarcompounds have already been applied in the clinic. For example certainnucleotide analogs have been effectively used for the inhibition of theretroviral reverse transcriptase in HIV therapy, as well as for otherviral infections, and in the treatment of certain cancers. As aproof-of-principle experiment we have demonstrated that addition of achain terminator nucleotide, the analog 2′,3′-dideoxy-UTP, effectivelyinhibits TUTase activity in vitro by preventing oilgo-U tail elongation.Thus, 2′,3′-dideoxy-UTP serves as a positive control nucleotide analogfor the in vitro HTS. Then, putative hits from this primary screen areevaluated through secondary screens that include dose curves, in vitroTUTase activity assays, and analyses of let-7 expression by q.RT-PCR oncompound-treated cells. The chemical HTS can be performed using theservices and chemical libraries, such as those available at theInstitute of Chemistry and Cell Biology (ICCB)-Longwood, an investigatorinitiated screening program that assists academic researchers incarrying out high-throughput screens of chemical libraries.

To evaluate the efficacy of small molecule inhibitors of the Lin28pathway as potential chemotherapeutics, identified compounds are testedfor their ability to inhibit proliferation, migration, and colonyformation of Lin28A-expressing human cancer cells. The validated smallmolecule inhibitors are examined for their ability to inhibitproliferation of Lin28A-expressing human cancer cell lines. Controlcells and compound-treated cells are counted daily using an automatedcounter for bright field cells. Expression of proliferation markers Ki67and phospho-Histone H3 can be measured as well. Decreased proliferationof cancer cells is expected in cells in which let-7 expression isrestored by chemical treatment.

The effects of hit compounds in colony formation and cell invasionassays are also examined, as described herein. For example, ColonyFormation Assay: T47D breast cancer cells and IGROV1 ovarian cancercells are treated with selected compounds for 24 hours. Triplicatesamples of 10⁵ cells from each treatment are mixed 4:1 (v/v) with 2.0%agarose in growth medium for a final concentration of 0.4% agarose. Thecell mixture is plated on top of a solidified layer of 0.5% agarose ingrowth medium. Cells are fed every 6 to 7 days with growth mediumcontaining 0.4% agarose. The number of colonies is counted after 20days. The experiment is repeated and the statistical significancecalculated using Student's t test. Invasion Assays: MDA-MB-231 and T47Dbreast cancer cells are treated with different compounds for 24 hours.Invasion of matrigel is conducted using standardized conditions withBDBioCoat growth factor reduced MATRIGEL invasion chambers (PharMingen).Assays are conducted per manufacturer's protocol, using 10% FBS aschemoattractant. Non-invading cells on the top-side of the membrane areremoved and invading cells are fixed and stained with DAPI, 16hours-post seeding. Such assays explored the distinct requirements forZcchc11 in Lin28A- and Lin28B-expressing cancer and established Zcch11as new therapeutic target in human cancers. The effects of Zcchc11inhibition on the tumorigenicity and invasiveness of MDA-MB-231 breastcancer cells (Lin28B-expressing cells) relative to T47D breast cancercells (Lin28A-expressing cells) were compared. Suppression of Zcchc11expression did not affect let-7a expression in MDA-MB-231 cells, but ledto 7-fold increase in mature let-7a levels in T47D cells.

Furthermore, Zcchc11 inhibition did not affect the tumorigenicity andinvasiveness of MDA-MB-231 cells, while it suppressed both the colonyformation ability and invasiveness of T47D cells. Zcchc11 inhibition hadsimilar effects on the tumor growth of these cell lines in xenografts.For these in vivo experiments, cells were injected subcutaneously in theright flank of athymic nude mice. Tumor growth was monitored every 5days. In Vivo Ready siRNAs (Ambion Inc.) were mixed with Invivofectamine2.0 liposomes (Ambion Inc.) and injected intra-peritoneal in a volume of1000 at a dose of 5 mg/kg. Specifically, Zcchc11 knockdown did notaffect tumor growth of MDA-MB-231, but it suppressed T47D tumor growth.Synthetic let-7a miRNA suppressed both MDA-MB-231 and T47D tumor growth.Also, in the tumors derived from MDA-MB-231 xenografts (day 30), let-7aexpression was not affected by inhibition of Zcchc11, while Lin28Bsuppression increased let-7a levels about 5-fold.

On the other hand, both Zcchc11 and Lin28A inhibition resulted inup-regulation of let-7a expression to similar levels in T47D-derivedtumors (day 30). In addition to the breast cancer cells, the effects ofZcchc11 inhibition were tested on tumor growth of several other (liver,lung, ovarian, melanoma, colon) cancer cell types. As above, Zcchc11inhibition blocked the growth of LIN28A-expressing tumors (Igrov1)(FIGS. 11A-11E) and did not affect the growth of Lin28B-expressingtumors (HepG2, H1299, SK_MEL_28, CaCO2). Lin28A and Lin28B inhibitionsuppressed the growth of the corresponding tumors. Taken together, thesedata suggest that Zcchc11 plays a role in the tumorigenicity andinvasiveness of Lin28A-expressing cancer cells. Specificity andToxicity: The effects of selected compounds on the growth and viabilityof cells that do not express Lin28A are examiend. For example, MCF-10A,non-tumorigenic breast epithelial cells that do not express Lin28A (orLin28B) are treated with these compounds to monitor the effect on thegrowth properties of these cells. MCF-10a cells are not expected to beinhibited by the compounds that selectively target the Lin28 pathway.This helps eliminate general cytotoxic compounds from analysis.Similarly, for compounds identified as TUTase inhibitors, they areexpected to phenocopy the effects of Zcchc11 depletion in breast cancercells. Therefore, these compounds should inhibit the growth of T47D, andIGROV1 cells (Lin28A-expressing) but not MDA-MB-231 cells(Lin28B-expressing).

Screen optimization is central to the success of HTS. The present datademonstrate the feasibility of this strategy. Additionally, for example,because two different promoters drive expression of the Firefly andRenilla Luciferase, one might replace one of the promoters so that bothLuciferases have the same kind of promoter to eliminate false positivesdue to differential affect of hits on each promoter. Additionally, todetect enhancement of let-7 activity, turnover of Renilla protein isrequired (detectable decrease in protein within the 48 hour incubationperiod) in order to observe changes in RLuc/Fluc ratio. Therefore t½ isa major determinant of screen dynamics and inhibitors of the Lin28pathway may be easier to find using a longer incubation times. Also,whether some of the ‘hit’ compounds are simply toxic, affect cellviability, influence cell proliferation, or lead to changes in thecell-cycle requires tesing. Whether any of these parameters causesdirect or indirect changes in RLuc/Fluc ratio imay be examined. Forexample, it has been reported that miRNA biogenesis may be influenced bycell density. Whether cell confluence changes the RLuc/Fluc ratio isrelevant when deciding on hit selection parameters and original cellseeding density. Z scores might not be the best or the sole parameter touse, and the inclusion of additional criteria for hit selection ispossible. This optimization is based on the data collected fromsecondary screens and may be essential for the development of the robustassay for HTS. As part of the compound validation process, to provideextra confidence in the hits, and to try to exclude off-target effects,additional compounds known to act in the same biological pathway may becompared.

Herein, the inventors demonstrate that Lin28A enhances repression ofpre-let-7 miRNA by recruiting a TUTase, specifically Zcchc11 and Zcchc6.Inhibition or depletion of such TUTase levels by Zcchc11 shRNA knockdownin a multiple human cancer cell lines which express Lin28 results in theincrease in levels of members of the mature let-7 family oftumor-suppressor miRs and a decrease in cell growth, colony formingcapacity, and tumor formation.

Accordingly, in discovering that Lin28A recruited TUTase is an inhibitorof miRNA processing and represses biogenesis of let-7 miRNA, whichrepression contributes to cancer, the inventors have discovered thatinhibition of Lin28A-recruited TUTase, e.g., Zcchc11 or Zcchc6, is auseful target for the treatment or prevention of cancer. Accordingly,one aspect of the present invention relates to a method to treat and/orprevent cancer by inhibition of Lin28A-recuited TUTase.

In some embodiments, the inhibitor agents of Lin28A-recruited TUTase canbe, for example, antibodies (polyclonal or monoclonal), neutralizingantibodies, antibody portions, fragments, analogs, variants orderivatives, peptides, proteins, peptide-mimetics, aptamers,oligonucleotides, hormones, small molecules, nucleic acids, nucleic acidanalogues, carbohydrates, or analogs, derviatives or variants thereof,that function to inactivate the nucleic acid and/or protein of the geneproducts identified herein, and those as yet unidentified. Nucleic acidsinclude, for example but not limited to, DNA, RNA, oligonucleotides,peptide nucleic acid (PNA), pseudo-complementary-PNA (pcPNA), lockednucleic acid (LNA), RNAi, microRNAi, siRNA, shRNA etc. The inhibitorscan be selected from a group of a chemical, small molecule, chemicalentity, nucleic acid sequences, nucleic acid analogues or protein orpolypeptide or analogue or fragment thereof. In some embodiments, thenucleic acid is DNA or RNA, and nucleic acid analogues, for example canbe PNA, pcPNA and LNA. A nucleic acid may be single or double stranded,and can be selected from a group comprising; nucleic acid encoding aprotein of interest, oligonucleotides, PNA, etc. Such nucleic acidsequences include, for example, but not limited to, nucleic acidsequence encoding proteins that act as transcriptional repressors,antisense molecules, ribozymes, small inhibitory nucleic acid sequences,for example but not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi),antisense oligonucleotides etc. A protein and/or peptide inhibitor orportion thereof, can be, for example, mutated proteins, therapeuticproteins and recombinant proteins. Proteins and peptides inhibitors canalso include for example; mutated proteins, genetically modifiedproteins, peptides, synthetic peptides, recombinant proteins, chimericproteins, antibodies, humanized proteins, humanized antibodies, chimericantibodies, modified proteins and fragments thereof.

As used herein, “gene silencing” or “gene silenced” in reference to anactivity of a RNAi molecule, for example a siRNA or miRNA refers to adecrease in the mRNA level in a cell for a target gene by at least about5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 99%, about 100% of themRNA level found in the cell without the presence of the miRNA or RNAinterference molecule. In one preferred embodiment, the mRNA levels aredecreased by at least about 70%, about 80%, about 90%, about 95%, about99%, about 100%.

As used herein, the term “RNAi” refers to any type of interfering RNA,including but are not limited to, siRNAi, shRNAi, endogenous microRNAand artificial microRNA. For instance, it includes sequences previouslyidentified as siRNA, regardless of the mechanism of down-streamprocessing of the RNA (i.e. although siRNAs are believed to have aspecific method of in vivo processing resulting in the cleavage of mRNA,such sequences can be incorporated into the vectors in the context ofthe flanking sequences described herein.

As used herein an “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a gene or target gene when the siRNA is present orexpressed in the same cell as the target gene, for example where atarget gene is Lin28A-recruited TUTase (e.g., Zcchc11 or Zcchc6). Thedouble stranded RNA siRNA can be formed by the complementary strands. Inone embodiment, a siRNA refers to a nucleic acid that can form a doublestranded siRNA. The sequence of the siRNA can correspond to the fulllength target gene, or a subsequence thereof. Typically, the siRNA is atleast about 15-50 nucleotides in length (e.g., each complementarysequence of the double stranded siRNA is about 15-50 nucleotides inlength, and the double stranded siRNA is about 15-50 base pairs inlength, preferably about 19-30 base nucleotides, preferably about 20-25nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length.

As used herein “shRNA” or “small hairpin RNA” (also called stem loop) isa type of siRNA. In one embodiment, these shRNAs are composed of ashort, e.g. about 19 to about 25 nucleotide, antisense strand, followedby a nucleotide loop of about 5 to about 9 nucleotides, and theanalogous sense strand. Alternatively, the sense strand can precede thenucleotide loop structure and the antisense strand can follow.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand (stem portion) that is linked on oneside by a region of predominantly single-stranded nucleotides (loopportion). The terms “hairpin” and “fold-back” structures are also usedherein to refer to stem-loop structures. Such structures are well knownin the art and the term is used consistently with its known meaning inthe art. The actual primary sequence of nucleotides within the stem-loopstructure is not critical to the practice of the invention as long asthe secondary structure is present. As is known in the art, thesecondary structure does not require exact base-pairing. Thus, the stemmay include one or more base mismatches. Alternatively, the base-pairingmay be exact, i.e. not include any mismatches. In some instances theprecursor microRNA molecule may include more than one stem-loopstructure. The multiple stem-loop structures may be linked to oneanother through a linker, such as, for example, a nucleic acid linker orby a microRNA flanking sequence or other molecule or some combinationthereof. The actual primary sequence of nucleotides within the stem-loopstructure is not critical as long as the secondary structure is present.As is known in the art, the secondary structure does not require exactbase-pairing. Thus, the stem may include one or more base mismatches.Alternatively, the base pairing may not include any mismatches.

As used herein, the term “let-7” refers to the nucleic acid encoding thelet-7 miRNA family members and homologues and variants thereof includingconservative substitutions, additions, and deletions therein notadversely affecting the structure or function. For example, let-7 refersto the nucleic acid encoding a let-7 family member from humans,including but not limited to, NCBI Accession Nos. AJ421724, AJ421725,AJ421726, AJ421727, AJ421728, AJ421729, AJ421730, AJ421731, AJ421732,and biologically active sequence variants of let-7, including alleles,and in vitro generated derivatives of let-7 that demonstrate let-7activity.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat are comprised of two strands. Double-stranded molecules includethose comprised of a single RNA molecule that doubles back on itself toform a two-stranded structure. For example, the stem loop structure ofthe progenitor molecules from which the single-stranded miRNA isderived, called the pre-miRNA (Bartel et al., 116 Cell 281 (2004)),comprises a dsRNA molecule.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and for the purposes of theinvention are limited to a minimum length of at least 20 amino acids.Oligopeptides, oligomers multimers, and the like, typically refer tolonger chains of amino acids and are also composed of linearly arrangedamino acids linked by peptide bonds, and whether produced biologically,recombinantly, or synthetically and whether composed of naturallyoccurring or non-naturally occurring amino acids, are included withinthis definition. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include co-translational(e.g., signal peptide cleavage) and post-translational modifications ofthe polypeptide, such as, for example, disulfide-bond formation,glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g.,cleavage by furins or metalloproteases), and the like. Furthermore, forpurposes of the present invention, a “polypeptide” refers to a proteinthat includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature as would be known to aperson in the art), to the native sequence, as long as the proteinmaintains the desired activity. These modifications can be deliberate,as through site-directed mutagenesis, or can be accidental, such asthrough mutations of hosts that produce the proteins, or errors due toPCR amplification or other recombinant DNA methods. Polypeptides orproteins are composed of linearly arranged amino acids linked by peptidebonds, but in contrast to peptides, has a well-defined conformation.Proteins, as opposed to peptides, generally consist of chains of 15 ormore amino acids. For the purposes of the present invention, the term“peptide” as used herein typically refers to a sequence of amino acidsof made up of a single chain of D- or L-amino acids or a mixture of D-and L-amino acids joined by peptide bonds. Generally, peptides containat least two amino acid residues and are less than about 15 amino acidsin length.

It will be appreciated that a protein or polypeptide often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, can be modified in a given polypeptide, eitherby natural processes such as glycosylation and other post-translationalmodifications, or by chemical modification techniques which are wellknown in the art. Known modifications which can be present in peptidesof the present invention include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of apolynucleotide or polynucleotide derivative, covalent attachment of alipid or lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formulation, gamma-c arboxylation, glycation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination.

The incorporation of non-natural amino acids, including syntheticnon-native amino acids, substituted amino acids, or one or more D-aminoacids into the peptides (or other components of the composition, withexception for protease recognition sequences) is desirable in certainsituations. D-amino acid-containing peptides exhibit increased stabilityin vitro or in vivo compared to L-amino acid-containing forms. Thus, theconstruction of peptides incorporating D-amino acids can be particularlyuseful when greater in vivo or intracellular stability is desired orrequired. More specifically, D-peptides are resistant to endogenouspeptidases and proteases, thereby providing better oral trans-epithelialand transdermal delivery of linked drugs and conjugates, improvedbioavailability of membrane-permanent complexes (see below for furtherdiscussion), and prolonged intravascular and interstitial lifetimes whensuch properties are desirable. The use of D-isomer peptides can alsoenhance transdermal and oral trans-epithelial delivery of linked drugsand other cargo molecules. Additionally, D-peptides cannot be processedefficiently for major histocompatibility complex class II-restrictedpresentation to T helper cells, and are therefore less likely to inducehumoral immune responses in the whole organism. Peptide conjugates cantherefore be constructed using, for example, D-isomer forms of cellpenetrating peptide sequences, L-isomer forms of cleavage sites, andD-isomer forms of therapeutic peptides. In some embodiments, apolypeptide inhibitor of Lin28A-recruited TUTase can be comprised of D-or L-amino acid residues, as use of naturally occurring L-amino acidresidues has the advantage that any break-down products should berelatively non-toxic to the cell or organism.

In yet a further embodiment, a polypeptide inhibitor of aLin28A-recruited TUTase can be a retro-inverso peptide. A “retro-inversopeptide” refers to a peptide with a reversal of the direction of thepeptide bond on at least one position, i.e., a reversal of the amino-and carboxy-termini with respect to the side chain of the amino acid.Thus, a retro-inverso analogue has reversed termini and reverseddirection of peptide bonds while approximately maintaining the topologyof the side chains as in the native peptide sequence. The retro-inversopeptide can contain L-amino acids or D-amino acids, or a mixture ofL-amino acids and D-amino acids, up to all of the amino acids being theD-isomer. Partial retro-inverso peptide analogues are polypeptides inwhich only part of the sequence is reversed and replaced withenantiomeric amino acid residues. Because the retro-inverted portion ofsuch an analogue has reversed amino and carboxyl termini, the amino acidresidues flanking the retro-inverted portion are replaced byside-chain-analogous .alpha.-substituted geminal-diaminomethanes andmalonates, respectively. Retro-inverso forms of cell penetratingpeptides have been found to work as efficiently in translocating acrossa membrane as the natural forms. See Bonelli et al., 24 Intl. J. Pept.Protein Res. 553 (1984); Verdini & Viscomi, 1 J. Chem. Soc. PerkinTrans. 697 (1985); U.S. Pat. No. 6,261,569. Processes for thesolid-phase synthesis of partial retro-inverso peptide analogues havebeen described (EP 97994-B).

The terms “homology”, “identity” and “similarity” refer to the degree ofsequence similarity between two peptides or between two optimallyaligned nucleic acid molecules. Homology and identity can each bedetermined by comparing a position in each sequence which can be alignedfor purposes of comparison. For example, it is based upon using astandard homology software in the default position, such as BLAST,version 2.2.14. When an equivalent position in the compared sequences isoccupied by the same base or amino acid, then the molecules areidentical at that position; when the equivalent site occupied by similaramino acid residues (e.g., similar in steric and/or electronic naturesuch as, for example conservative amino acid substitutions), then themolecules can be referred to as homologous (similar) at that position.Expression as a percentage of homology/similarity or identity refers toa function of the number of similar or identical amino acids atpositions shared by the compared sequences, respectfully. A sequencewhich is “unrelated” or “non-homologous” shares less than 40% identity,though preferably less than 25% identity with the sequences as disclosedherein.

As used herein, the term “sequence identity” means that twopolynucleotide or amino acid sequences are identical (i.e., on anucleotide-by-nucleotide or residue-by-residue basis) over thecomparison window. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical nucleic acid base (e.g., A, T. C, G. U. or I) or residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the comparison window (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

As used herein, the terms “homologous” or “homologues” are usedinterchangeably, and when used to describe a polynucleotide orpolypeptide, indicates that two polynucleotides or polypeptides, ordesignated sequences thereof, when optimally aligned and compared, forexample using BLAST, version 2.2.14 with default parameters for analignment (see herein) are identical, with appropriate nucleotideinsertions or deletions or amino-acid insertions or deletions, in atleast 70% of the nucleotides, usually from about 75% to 99%, and morepreferably at least about 98 to 99% of the nucleotides. The term“homolog” or “homologous” as used herein also refers to homology withrespect to structure and/or function. With respect to sequence homology,sequences are homologs if they are at least 50%, at least 60% at least70%, at least 80%, at least 90%, at least 95% identical, at least 97%identical, or at least 99% identical. Determination of homologs of thegenes or peptides of the present invention can be easily ascertained bythe skilled artisan. The term “substantially homologous” refers tosequences that are at least 90%, at least 95% identical, at least 96%,identical at least 97% identical, at least 98% identical or at least 99%identical.

Homologous sequences can be the same functional gene in differentspecies. Determination of homologs of the genes or peptides of thepresent invention can be easily ascertained by the skilled artisan. Forsequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, forexample, by a local homology algorithm (Smith & Waterman, 2 Adv. Appl.Math. 482 (1981)); by the homology alignment algorithm (Needleman &Wunsch, 48 J. Mol. Biol. 443 (1970)); by the search for similaritymethod (Pearson & Lipman, 85 PNAS2444 (1988)); by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.); by PILEUP, a simplification ofthe progressive alignment method (Feng & Doolittle, 25 J. Mol. Evol. 351(1987)); by BLAST algorithm (Altschul et al., 215 J. Mol. Biol. 403(1990)); or by visual inspection (see generally Ausubel et al. (eds.),Curr. Prot. Molec. Biol. (4th ed., John Wiley & Sons, New York, 1999)).

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the naturally occurring polypeptide or nucleic acid byone or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or morespecific functions or biological activities of the naturally occurringmolecule. Amino acid substitutions include alterations in which an aminoacid is replaced with a different naturally-occurring or anon-conventional amino acid residue. Such substitutions may beclassified as “conservative”, in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality or size. Substitutions encompassed by the presentinvention may also be “non conservative”, in which an amino acid residuewhich is present in a peptide is substituted with an amino acid havingdifferent properties, such as naturally-occurring amino acid from adifferent group (e.g., substituting a charged or hydrophobic amino; acidwith alanine), or alternatively, in which a naturally-occurring aminoacid is substituted with a non-conventional amino acid. In someembodiments amino acid substitutions are conservative. Also encompassedwithin the term variant when used with reference to a polynucleotide orpolypeptide, refers to a polynucleotide or polypeptide that can vary inprimary, secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide).

Variants can be naturally-occurring, synthetic, recombinant, orchemically modified polynucleotides or polypeptides isolated orgenerated using methods well known in the art. Variants can includeconservative or non-conservative amino acid changes, as described below.Polynucleotide changes can result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. Variants can also include insertions,deletions or substitutions of amino acids, including insertions andsubstitutions of amino acids and other molecules) that do not normallyoccur in the peptide sequence that is the basis of the variant, forexample but not limited to insertion of ornithine which do not normallyoccur in human proteins. The term “conservative substitution,” whendescribing a polypeptide, refers to a change in the amino acidcomposition of the polypeptide that does not substantially alter thepolypeptide's activity. For example, a conservative substitution refersto substituting an amino acid residue for a different amino acid residuethat has similar chemical properties. Conservative amino acidsubstitutions include replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, or a threonine with a serine.“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the BBB).Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, the following six groupseach contain amino acids that are conservative substitutions for oneanother: (1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid(D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine(R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). See alsoCreighton, PROTEINS (W. H. Freeman & Company, 1984). In someembodiments, individual substitutions, deletions or additions thatalter, add or delete a single amino acid or a small percentage of aminoacids can also be considered “conservative substitutions” is the changedoes not reduce the activity of the peptide (i.e., the ability of aLinA-recruited TUTase polypeptide to process the maturation of miRNA).Insertions or deletions are typically in the range of about one to fiveamino acids. The choice of conservative amino acids may be selectedbased on the location of the amino acid to be substituted in thepeptide, for example if the amino acid is on the exterior of the peptideand expose to solvents, or on the interior and not exposed to solvents.

In alternative embodiments, one can select the amino acid which willsubstitute an existing amino acid based on the location of the existingamino acid, i.e., its exposure to solvents (i.e., if the amino acid isexposed to solvents or is present on the outer surface of the peptide orpolypeptide as compared to internally localized amino acids not exposedto solvents). Selection of such conservative amino acid substitutionsare well known in the art. Accordingly, one can select conservativeamino acid substitutions suitable for amino acids on the exterior of aprotein or peptide (i.e. amino acids exposed to a solvent), for example,but not limited to, the following substitutions can be used:substitution of Y with F, T with S or K, P with A, E with D or Q, N withD or G, R with K, G with N or A, T with S or K, D with N or E, I with Lor V, F with Y, S with T or A, R with K, G with N or A, K with R, A withS, K or P.

The term “functional” when used in conjunction with “derivative” or“variant” refers to a molecule such as a protein which possess abiological activity (either functional or structural) that issubstantially similar to a biological activity of the entity or moleculeits is a functional derivative or functional variant thereof. The termfunctional derivative is intended to include the fragments, analogues orchemical derivatives of a molecule. A molecule is said to be“substantially similar” to another molecule if both molecules havesubstantially similar structures or if both molecules possess a similarbiological activity, for example if both molecules are able to deliver atarget antigen to the cytosol of a cell in the absence of PA and withoutbeing fused to the target antigen. Thus, provided that two moleculespossess a similar activity, they are considered variants and areencompassed for use as disclosed herein, even if the structure of one ofthe molecules not found in the other, or if the sequence of amino acidresidues is not identical. Thus, provided that two molecules possess asimilar biological activity, they are considered variants as that termis used herein even if the structure of one of the molecules not foundin the other, or if the sequence of amino acid residues is notidentical.

The term “disease” or “disorder” is used interchangeably herein, refersto any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,amlady, disorder, sickness, illness, complaint, inderdisposion,affection.

The term “malignancy” and “cancer” are used interchangeably herein,refers to diseases that are characterized by uncontrolled, abnormalgrowth of cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. The term“malignancy” or “cancer” are used interchangeably herein and refers toany disease of an organ or tissue in mammals characterized by poorlycontrolled or uncontrolled multiplication of normal or abnormal cells inthat tissue and its effect on the body as a whole. Cancer diseaseswithin the scope of the definition comprise benign neoplasms,dysplasias, hyperplasias as well as neoplasms showing metastatic growthor any other transformations like e.g. leukoplakias which often precedea breakout of cancer. The term “tumor” or “tumor cell” are usedinterchangeably herein, refers to the tissue mass or tissue type of cellthat is undergoing abnormal proliferation.

A “cancer cell” refers to a cancerous, pre-cancerous or transformedcell, either in vivo, ex vivo, and in tissue culture, that hasspontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation/cancer is associated with,e.g., morphological changes, immortalization of cells, aberrant growthcontrol, foci formation, anchorage dependence, proliferation,malignancy, contact inhibition and density limitation of growth, growthfactor or serum dependence, tumor specific markers levels, invasiveness,tumor growth or suppression in suitable animal hosts such as nude mice,and the like, in vitro, in vivo, and ex vivo. See also Freshney, CultureAnimal Cells: A Man. Basic Tech. (3rd ed., 1994).

The term “biological sample” as used herein may mean a sample ofbiological tissue or fluid that comprises nucleic acids. Such samplesinclude, but are not limited to, tissue isolated from animals.Biological samples may also include sections of tissues such as biopsyand autopsy samples, frozen sections taken for histologic purposes,blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin.Biological samples also include explants and primary and/or transformedcell cultures derived from patient tissues. A biological sample may beprovided by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. As used herein, theterm “biological sample” also refers to a cell or population of cells ora quantity of tissue or fluid from a subject. “Biological sample” canalso refer to cells or tissue analyzed in vivo, i.e., without removalfrom the subject. The term “tissue” is intended to include intact cells,blood, blood preparations such as plasma and serum, bones, joints,muscles, smooth muscles, and organs.

The terms “patient”, “subject” and “individual” are used interchangeablyherein, and refer to an animal, particularly a human, to whom treatment,including prophylactic treatment is provided. The term “subject” as usedherein refers to human and non-human animals, such as apes, monkeys,horses, cattle, sheep, goats, dogs, cats, rabbits, guinea pigs, rats,and mice. In one embodiment, the subject is human. In anotherembodiment, the subject is an experimental animal or animal substituteas a disease model.

The term “gene” used herein can be a genomic gene comprisingtranscriptional and/or translational regulatory sequences and/or acoding region and/or non-translated sequences (e.g., introns, 5′- and3′-untranslated sequences and regulatory sequences). The coding regionof a gene can be a nucleotide sequence coding for an amino acid sequenceor a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA andantisense RNA. A gene can also be an mRNA or cDNA corresponding to thecoding regions (e.g., exons and miRNA) optionally comprising 5′- or 3′untranslated sequences linked thereto. A gene can also be an amplifiednucleic acid molecule produced in vitro comprising all or a part of thecoding region and/or 5′- or 3′-untranslated sequences linked thereto.

The term “nucleic acid” or “oligonucleotide” or “polynucleotide” usedherein can mean at least two nucleotides covalently linked together. Aswill be appreciated by those in the art, the depiction of a singlestrand also defines the sequence of the complementary strand. Thus, anucleic acid also encompasses the complementary strand of a depictedsingle strand. As will also be appreciated by those in the art, manyvariants of a nucleic acid can be used for the same purpose as a givennucleic acid. Thus, a nucleic acid also encompasses substantiallyidentical nucleic acids and complements thereof. As will also beappreciated by those in the art, a single strand provides a probe for aprobe that can hybridize to the target sequence under stringenthybridization conditions. Thus, a nucleic acid also encompasses a probethat hybridizes to a “target” under stringent hybridization conditions.Nucleic acids can be single stranded or double stranded, or can containportions of both double stranded and single stranded sequence. Thenucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine. Nucleic acids can be obtained by chemical synthesismethods or by recombinant methods.

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs can be included that can have at least onedifferent linkage, e.g., phosphoramidate, phosphorothioate,phosphorodithioate, or O-methylphosphoroamidite linkages and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. No. 5,235,033 and U.S.Pat. No. 5,034,506. Nucleic acids containing one or more non-naturallyoccurring or modified nucleotides are also included within onedefinition of nucleic acids. The modified nucleotide analog can belocated for example at the 5′-end and/or the 3′-end of the nucleic acidmolecule. Representative examples of nucleotide analogs can be selectedfrom sugar- or backbone-modified ribonucleotides. It should be noted,however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.,8-bromo guanosine; deaza nucleotides, e.g., 7 deaza-adenosine; 0- andN-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. The 2′OH-group can be replaced by a group selected from H, OR, Rhalo, SH, SR,NH₂, NHR, NR₂ or CN, wherein R is C1-C6 alkyl, alkenyl or alkynyl andhalo is F, Cl, Br, or I. Modifications of the ribose-phosphate backbonecan be done for a variety of reasons, e.g., to increase the stabilityand half-life of such molecules in physiological environments or asprobes on a biochip. Mixtures of naturally occurring nucleic acids andanalogs can be made; alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occurring nucleic acids and analogscan be made.

The term “agent” refers to any entity which is normally absent or notpresent at the levels being administered, in the cell. Agent may beselected from a group comprising; chemicals; small molecules; nucleicacid sequences; nucleic acid analogues; proteins; peptides; aptamers;antibodies; or fragments thereof. A nucleic acid sequence may be RNA orDNA, and may be single or double stranded, and can be selected from agroup comprising; nucleic acid encoding a protein of interest,oligonucleotides, nucleic acid analogues, for example peptide-nucleicacid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid(LNA), etc. Such nucleic acid sequences include, for example, but notlimited to, nucleic acid sequence encoding proteins, for example thatact as transcriptional repressors, antisense molecules, ribozymes, smallinhibitory nucleic acid sequences, for example but not limited to RNAi,shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc. Aprotein and/or peptide or fragment thereof can be any protein ofinterest, for example, but not limited to; mutated proteins; therapeuticproteins; truncated proteins, wherein the protein is normally absent orexpressed at lower levels in the cell. Proteins can also be selectedfrom a group comprising; mutated proteins, genetically engineeredproteins, peptides, synthetic peptides, recombinant proteins, chimericproteins, antibodies, midibodies, tribodies, humanized proteins,humanized antibodies, chimeric antibodies, modified proteins andfragments thereof. The agent may be applied to the media, where itcontacts the cell and induces its effects. Alternatively, the agent maybe intracellular within the cell as a result of introduction of thenucleic acid sequence into the cell and its transcription resulting inthe production of the nucleic acid and/or protein environmental stimuliwithin the cell. In some embodiments, the agent is any chemical, entityor moiety, including without limitation synthetic andnaturally-occurring non-proteinaceous entities. In certain embodimentsthe agent is a small molecule having a chemical moiety. For example,chemical moieties included unsubstituted or substituted alkyl, aromatic,or heterocyclyl moieties including macrolides, leptomycins and relatednatural products or analogues thereof. Agents can be known to have adesired activity and/or property, or can be selected from a library ofdiverse compounds.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a condition, disease or disorderassociated with inappropriate proliferation, for example cancer. As usedherein, the term treating is used to refer to the reduction of a symptomand/or a biochemical marker of in appropriate proliferation, for examplea reduction in at lease one biochemical marker of cancer by at least10%. For example, a reduction in a biochemical marker of cancer, forexample a reduction in, as an illustrative example only, at least one ofthe following biomarkers; CD44, telomerase, TGF-α, TGF-α, erbB-2,erbB-3, MUC1, MUC2, MUC5, CK20, PSA, CA125 or FOBT by at least 10%; or areduction in the rate of proliferation of the cancer cells by 10%, wouldbe considered effective treatments by the methods as disclosed herein.As alternative examples, a reduction in a symptom of cancer, forexample, a slowing of the rate of growth of the cancer by 10%, or acessation of the increase in tumor size, or a reduction in the size of atumor by 10% or a reduction in the tumor spread (i.e. tumor metastasis)by 10% would also be considered as affective treatments by the methodsas disclosed herein.

The term “effective amount” as used herein refers to the amount of atleast one agent of pharmaceutical composition to reduce or stop at leastone symptom of the abnormal proliferation, for example a symptom of acancer or malignancy. For example, an effective amount using the methodsas disclosed herein would be considered as the amount sufficient toreduce a symptom of the abnormal proliferation, for example at least onesymptom of a cancer or malignancy by at least 10%. An effective amountas used herein would also include an amount sufficient to prevent ordelay the development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slow the progression ofa symptom of the disease), or reverse a symptom of the disease.

As used herein, the terms “administering,” and “introducing” are usedinterchangeably and refer to the placement of the agents into a subjectby a method or route which results in at least partial localization ofthe agents at a desired site. The compounds of the present invention canbe administered by any appropriate route which results in an effectivetreatment in the subject.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” means a pharmaceuticallyacceptable material, composition or vehicle, such as a liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting the subject agents from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorscapable of directing the expression of genes and/or nucleic acidsequence to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome. Other expression vectors can be used indifferent embodiments of the invention, for example, but are not limitedto, plasmids, episomes, bacteriophages or viral vectors, and suchvectors can integrate into the host's genome or replicate autonomouslyin the particular cell. Other forms of expression vectors known by thoseskilled in the art which serve the equivalent functions can also beused. Expression vectors comprise expression vectors for stable ortransient expression encoding the DNA. A vector can be a plasmid,bacteriophage, bacterial artificial chromosome or yeast artificialchromosome. A vector can be a DNA or RNA vector. A vector can be eithera self replicating extrachromosomal vector or a vector which integrateinto a host genome.

The term “gene product(s)” as used herein refers to include RNAtranscribed from a gene, or a polypeptide encoded by a gene ortranslated from RNA.

The term “inhibition” or “inhibit” when referring to the gene expressionand/or activity or protein of a TUTase recruited by Lin28A, such asZcchc11 or Zcchc6, or a functional domain thereof, refers to a reductionor prevention in the level of its function or a reduction of its geneexpression product.

In some embodiments, agents that inhibit TUTases recruited by Lin28 arenucleic acids. Nucleic acid inhibitors of Lin28-recruited TUTases arefor example, but not are limited to, RNA interference-inducingmolecules, for example but are not limited to siRNA, dsRNA, stRNA, shRNAand modified versions thereof, where the RNA interference moleculesilences the gene expression of a TUTase such as Zcchc11 or Zcchc6. Insome embodiments, the nucleic acid inhibitor is an anti-senseoligonucleic acid, or a nucleic acid analogue, for example but are notlimited to DNA, RNA, peptide-nucleic acid (PNA), pseudo-complementaryPNA (pc-PNA), or locked nucleic acid (LNA) and the like. In alternativeembodiments, the nucleic acid is DNA or RNA, and nucleic acid analogues,for example PNA, pcPNA and LNA. A nucleic acid can be single or doublestranded, and can be selected from a group comprising nucleic acidencoding a protein of interest, oligonucleotides, PNA, etc. Such nucleicacid sequences include, for example, but are not limited to, nucleicacid sequence encoding proteins that act as transcriptional repressors,antisense molecules, ribozymes, small inhibitory nucleic acid sequences,for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi(mRNAi), antisense oligonucleotides, etc. In general, RNA interferencetechnology is well known in the art, as are methods of delivering RNAinterfering agents. See, e.g., U.S. Patent Pub. No. 2010/0221226.

In some embodiments, the method as disclosed herein are useful for thetreatment of any disease or disorder characterized by lack or reducedexpression of tumor suppressor miRNAs, for example but not limited tolet-7 family miRNA, that are mediated by miRNA-binding protein-recruited3′ terminal uridylyl transferases (TUTases), such as Zcchc11 or Zcchc6or a functional domain thereof.

In one embodiment, a pharmaceutical composition as disclosed hereincomprises at least one agent which is an inhibitor of a Lin28A-recuitedTUTase can be administered for treatment or prevention of breast cancer.In some embodiments, the pharmaceutical composition as disclosed hereinwhich comprises at least one agent inhibitor of Lin28A-recruited TUTasecan be administered for treatment or prevention of, for example but notlimited to, breast cancer or prostate cancer.

In addition, the agents and pharmaceutical compositions as disclosedherein comprising inhibitors of a Lin28A-recruited TUTase can also beused for prophylactic treatment of cancer. There are hereditaryconditions and/or environmental situations (e.g., exposure tocarcinogens) known in the art that predispose an individual todeveloping cancers, or subjects identified to have increased expressionof Lin28A as compared to a reference sample. Under these circumstances,it may be beneficial to treat these individuals with therapeuticallyeffective doses of an agent which inhibit Lin28A-TUTase, such as Zcchc11or Zcchc6, to reduce the risk of developing cancers. Determination ofLin28A-associated risk is known in the art, as is monitoring Lin28levels in Lin28-associated cancers. See U.S. Patent Pub. No.2010/0221266.

In some embodiments, the agents and pharmaceutical compositions asdisclosed herein comprising at least one inhibitor of Lin28A-recruitedTUTase can be administered in therapeutically effective dosages alone orin combination with at least one other adjuvant cancer therapy such assurgery, chemotherapy, radiotherapy, thermotherapy, immunotherapy,hormone therapy, anti-cancer agent or laser therapy, to provide abeneficial effect, e.g. reducing tumor size, slowing rate of tumorgrowth, reducing cell proliferation of the tumor, promoting cancer celldeath, inhibiting angiogenesis, inhibiting metastasis, or otherwiseimproving overall clinical condition, without necessarily eradicatingthe cancer.

The term “chemotherapeutic agent” or “chemotherapy agent” are usedinterchangeably herein and refers to an agent that can be used in thetreatment of cancers and neoplasms, for example brain cancers andgliomas and that is capable of treating such a disorder. In someembodiments, a chemotherapeutic agent can be in the form of a prodrugwhich can be activated to a cytotoxic form. Chemotherapeutic agents arecommonly known by persons of ordinary skill in the art and areencompassed for use in the present invention. For example,chemotherapeutic drugs for the treatment of tumors and gliomas include,but are not limited to: temozolomide (Temodar), procarbazine (Matulane),and lomustine (CCNU). Chemotherapy given intravenously (by IV, vianeedle inserted into a vein) includes vincristine (Oncovin or VincasarPFS), cisplatin (Platinol), carmustine (BCNU, BiCNU), and carboplatin(Paraplatin), Mexotrexate (Rheumatrex or Trexall), irinotecan (CPT-11);erlotinib; oxalipatin; anthracyclins-idarubicin and daunorubicin;doxorubicin; alkylating agents such as melphalan and chlorambucil;cis-platinum, methotrexate, and alkaloids such as vindesine andvinblastine.

In another embodiment, the present invention encompasses combinationtherapy in which subjects identified as having, or increased risk ofdeveloping cancer by having increased levels of Lin28A protein orexpression as compared to a reference level using the methods asdisclosed herein are administered an anti-cancer combination therapywhere combinations of anti-cancer agents are used are used incombination with cytostatic agents, anti-VEGF and/or p53 reactivationagent. A cytostatic agent is any agent capable of inhibiting orsuppressing cellular growth and multiplication. Examples of cytostaticagents used in the treatment of cancer are paclitaxel, 5-fluorouracil,5-fluorouridine, mitomycin-C, doxorubicin, and zotarolimus. Other cancertherapeutics include inhibitors of matrix metalloproteinases such asmarimastat, growth factor antagonists, signal transduction inhibitorsand protein kinase C inhibitors.

The compositions as disclosed herein used in connection with thetreatment methods of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual subject, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including, but not limited to,improved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

An agent that inhibits a TUTas recruited by Lin28A can be used as amedicament or used to formulate a pharmaceutical composition with one ormore of the utilities disclosed herein. In some embodiments, an agentwhich inhibits Lin28A-recruited TUTase can be administered in vitro tocells in culture, in vivo to cells in the body, or ex vivo to cellsoutside of a subject that can later be returned to the body of the sameindividual or another. Such cells can be disaggregated or provided assolid tissue.

In some embodiments, an agent that inhibits Lin28A-recruited TUTase canbe used to produce a medicament or other pharmaceutical compositions.Use of the compositions as disclosed herein comprising an agentinhibiting Lin28-recruited TUTase can further comprise apharmaceutically acceptable carrier and/or additional components usefulfor delivering the composition to a subject. Such pharmaceuticallyacceptable carrier and/or additional components are well known in theart. Addition of such carriers and other components to the agents asdisclosed herein is well within the level of skill in this art.

Suitable choices in amounts and timing of doses, formulation, and routesof administration can be made with the goals of achieving a favorableresponse in the subject with cancer, for example a subject with canceror a subject at risk thereof (i.e., efficacy), and avoiding unduetoxicity or other harm thereto (i.e., safety). Therefore, “effective”refers to such choices that involve routine manipulation of conditionsto achieve a desired effect.

The embodiments of the invention described herein are further describedby the following numbered paragraphs:

1. A composition for inhibiting Lin28A-mediated let-7 repressioncomprising an agent that inhibits the interaction of Lin28A with 3′terminal uridylyl transferase (TUTase) or a functional portion thereof.

2. The composition of paragraph 1, wherein the TUTase is Zcchc11 orZcchc6.

3. A method of inhibiting Lin28A-mediated let-7 repression in a cellcomprising contacting said cell with an inhibitor of TUTase.

4. A method of increasing the expression level of let-7 in a tissue in asubject comprising administering a therapeutically effective amount ofan inhibitor of a Lin28A-recruited TUTase and a pharmaceuticallyacceptable carrier, whereby the amount of let-7 in the tissue isincreased in the presence of the inhibitor relative to in the absence ofthe inhibitor.

5. A system for high throughput screening of agents that derepress let-7expression comprising: a recombinant functional domain of TUTase Zcchc11or Zcchc6; a recombinant functional domain of Lin28A; a let-7 miRNAprecursor; a means for monitoring 3′ terminal uridylyl transferaseactivity; wherein an agent that derepresses let-7 expression inhibitsTUTase activity compared with an agent that does not derepress let-7expression.

6. A method of treating of a Lin28A-expressing cancer in a subject inneed thereof, comprising administering a therapeutically effectiveamount of an inhibitor of a TUTase and a pharmaceutically acceptablecarrier, whereby the growth of the Lin28A-expressing cancer is reducedin the presence of the inhibitor.

7. A method of treatment of cancer in a subject in need thereof, themethod comprising measuring the level of expression of Lin28A in acancerous tissue obtained from the subject; and administering atherapeutically effective amount of an inhibitor of a TUTase to thesubject when the cancerous tissue is positive for Lin28A expression.

8. A method of inhibiting tumor growth in a subject in need thereofcomprising administering a therapeutically effective amount of aninhibitor of a TUTase and a pharmaceutically acceptable carrier, wherebytumor growth is inhibited in the presence of the inhibitor.

9. A method of preventing tumor or cancer metastasis in a subject inneed thereof comprising administering a therapeutically effective amountof an inhibitor of a TUTase and a pharmaceutically acceptable carrier,whereby tumor or cancer metastasis is inhibited in the presence of theinhibitor.

10. The method of paragraph 8 or 9, wherein the tumor or cancer in thesubject expresses Lin28A.

11. The method of paragraph 8 or 9, further comprising measuring thelevel of expression of Lin28A in a in a sample of cancerous tissueobtained from the subject prior to administering the TUTase inhibitor.

12. The method of any one of paragraphs 6-11, wherein the TUTase isZcchc11 or Zcchc6.

13. The method of any one of paragraphs 6-12, wherein the inhibitorinhibits the expression of Zcchc11 or Zcchc6.

14. The method of any one of paragraphs 6-13, wherein the inhibitorinhibits the activity of Zcchc11 or Zcchc6.

15. The method of any one of paragraphs 6-14, wherein the inhibitor isselected from the group consisting of an antibody or a portion,derivative, analog, fragment or variant thereof, a RNA interferencemolecule, a small molecule, a peptide and an aptamer.

16. The method of paragraph 15, wherein the inhibitor is a RNAinterference molecule.

17. The method of paragraph 16, wherein the RNA interference molecule isa short-hairpin RNA.

18. The method of paragraph 17, wherein the RNA interference molecule isa short-hairpin RNA directed specifically against the Zcchc11 gene orthe Zcchc6 gene.

19. The method of any one of paragraphs 6-18, wherein the cancer iscolon, breast, ovarian, or prostate cancer.

20. A method of increasing the expression level of let-7a in a tissue ina subject comprising administering a therapeutically effective amount ofan inhibitor of a TUTase and a pharmaceutically acceptable carrier,whereby the amount of let-7a in the tissue is increased in the presenceof the inhibitor relative to in the absence of the inhibitor.

21. The method of paragraph 20, wherein the tissue is colon, prostate,ovarian, or breast tissue.

22. The method of paragraph 20, wherein the subject is diagnosed withcancer.

23. The method of paragraph 21, wherein the tissue is a canceroustissue.

24. The method of any one of paragraphs 20-23, wherein the tissue in thesubject expresses Lin28A.

25. The method of paragraph 20 further comprising measuring the level ofexpression of Lin28A in a sample of the tissue obtained from the subjectprior to administering the inhibitor.

26. The method of any one of paragraphs 20-25, wherein the TUTase isZcchc11 or Zcchc6.

27. The method of any one of paragraphs 20-26, wherein the inhibitorinhibits the expression of Zcchc11 or Zcchc6.

28. The method of any one of paragraphs 20-27, wherein the inhibitorinhibits the activity of Zcchc11 or Zcchc6.

29. The method of any one of paragraphs 20-28, wherein the inhibitor isselected from the group consisting of an antibody, a portion of anantibody, a RNA interference molecule, a small molecule, a peptide, andan aptamer.

30. The method of paragraph 29, wherein the inhibitor is a RNAinterference molecule.

31. The method of paragraph 30, wherein the RNA interference molecule isa short-hairpin RNA.

32. The method of paragraph 25, wherein the RNA interference molecule isa short-hairpin RNA directed specifically against the Zcchc11 gene.

33. The method of paragraph 25, wherein the RNA interference molecule isa short-hairpin RNA directed specifically against the Zcchc6 gene.

34. The method of any one of paragraphs 20-33, wherein the expressionlevel of let-7a level in the tissue is increased by at least 2-fold.

35. The method of any one of paragraphs 20-33, wherein the expressionlevel of let-7a level in the tissue is increased by about 2- to about10-fold.

EXAMPLES Example 1. Characterization of the Molecular Mechanisms of theLin28/Let-7 Axis

Cloning:

Myc-Lin28A and -Lin28B were cloned into pBK-EF1. Lin28A, Lin28B, andLin28BDNLS#1 were cloned into pFLAG-CMV2 vector (Sigma). Lin28BDNoLSDNLS#1 was generated by site-directed mutagenesis using the QuickChange kit (Stratagene). Lin28A, Lin28B, Lin28BDNLS#1, and Lin28BDNoLSDNLS#1 were cloned into CT-GFP-Topo (Invitrogen). NLS#1 and NLS#2oligos were annealed before ligating into CT-GFP-Topo. N-terminalCherry-DGCR8 fusion construct was generated by subcloning Cherry cDNAinto p3×FLAG-CMV14-DGCR8. Gregory et al., 2004. Lin28A and Lin28B weresubcloned into Pet21 for His-tagged recombinant protein expression.Pri-let-7g was previously reported. Viswanathan et al., 2008). Cloningprimers are listed in Table 1:

TABLE 1 Primers Cloning to pBK-EF1 Myc-Lin28A  TGCAGACCGGTGAGCAGAAACTCATAAGCGAAG AgeI For AGGACCTGGGCTCCGTGT CCAACCAGMyc-Lin28A  GACATGAAGCTTTCAATTCTGTGCCTCCGGGAG HindIII Rev Myc-Lin28B TGCAGACCGGTGAGCAGAAACTCATAAGCGAAG AgeI For AGGACCTGGCCGAAGGCG GGGCTAGMyc-Lin28B  TACGATTCTAGATTATGTCTTTTTCCTTTTTTG XbaI Rev AACTGAAGGC Myc-AGTCTATCTAGATTAGCTTTGCTCTTCTGGTGC Lin28BΔNLS#1  RevSite-directed mutagenesis Lin28B NoLS Lin28B Mut1 GACACTACAGGGAAGAGGACCAGGGGGAGATAG For Lin28B Mut1 CTATCTCCCCCTGGTCCTCTTCCCTGTAGTGTC Rev Lin28B Mut2 GGAAGTGAAAGAGGACCCGGAGGGAAGACACTA For Lin28B Mut2 TAGTGTCTTCCCTCCGGGTCCTCTTTCACTTCC Rev Cloning to pFlag-CMV2 Lin28A XbaI TATCGATCTAGAGGCTCCGTGTCCAACCAGCAG For Lin28A BamHI TATCGAGGATCCTTAATTCTGTGCCTCCGGGAG Rev CAGGG Lin28B XbaI TATCGATCTAGAGCCGAAGGCGGGGCTAGCAAA For G Lin28B BamHI CTCGCAGGATCCTTATGTCTTTTTCCTTTTTTG Rev AACTG Lin28BΔNLS#1 CGTCAGGGATCCTTAGCTTTGCTCTTCTGGTGC BamHI RevCloning Primers for CT-GFP-Topo Lin28A For GCCGCCATGGGCTCCGTGTCCAACCAGCLin28A Rev CATTCTGTGCCTCCGGGAGCAG Lin28B For GCCGCCATGGCCGAAGGCGGGGCTAGCLin28B Rev CTGTCTTTTTCCTTTTTTGAACTG Lin28BΔNLS#1  AAACTGAAGGCCCCTTTTTGCRev NLS#1 For GCCGCCATGAAAAAGGGGCCTTCAGTTCAAAAA AGGAAAAAGACAGA NLS#1 RevTCTGTCTTTTTCCTTTTTTGAACTGAAGGCCCC TTTTTCATGGCGGCA NLS#2 ForGCCGCCATGGGAAGAAGACCCAAAGGGAAGACA CTACAGAAAAGAAAACCAAAGGA NLS#2 RevCCTTTGGTTTTCTTTTCTGTAGTGTCTTCCCTT TGGGTCTTCTTCCCATGGCGGCACloning to Pet21a(+) His-Lin28A- GGATCCcatcatcaccatcaccacGGCTCCGTGBamHI For TCCAACCAGCAG Lin28A-NotI  GCGGCCGCTTACAGTTTGCGTACCAATAAG RevHis-Lin28B- GGATCCcatcatcaccatcaccacGCCGAAGGC BamHI For GGGGCTAGCAAAGLin28B-NotI  TTATGTCTTTTTCCTTTTTTGAAC Rev q.RT-PCR Primers hsa-pri-let-AGCGCTCCGTTTCCTTTT 7g For hsa-pri-let- CCCCACTTGGCAGCTG 7g Revhsa-pri-let- CCTGGATGTTCTCTTCACTG 7a-1 For hsa-pri-let-GCCTGGATGCAGACTTTTCT 7a-1 For hsa-pri-mir- GCTTATCAGACTGATGTTGACTG21 For hsa-pri-mir- CAGCCCATCGACTGGTG 21 Rev U6 For CTCGCTTCGGCAGCACAU6 Rev AACGCTTCACGAATTTGCGT Lin28A For AAGCGCAGATCAAAAGGAGA Lin28A RevCTGATGCTCTGGCAGAAGTG Lin28B For TGATAAACCGAGAGGGAAGC Lin28B RevTGTGAATTCCACTGGTTCTCC GAPDH For ATGTTCGTCATGGGTGTGAA GAPDH RevGGTGCTAAGCAGTTGGTGGT

TABLE 2 shRNAs used for knockdown Control ACCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTC shRNA#1TTCATCTTGTTGTTTTTGAATTC Control ACCGGGCCCGCAAGCTGACCCTGAAGTTCATTCAAGAGATG shRNA#2AACTTCAGGgTCAGCTTGCTTTTTGAATTC Control ACCGGGTCGGCTTACGGCGGTGATTTCTCGAGAAATCACCG shRNA#3CCGTAAGCCGACTTTTTGAATTC Zcchc11 ACCGGGTCAGTTACATTCAGCAGAAACTCGAGTTTCTGCTG shRNA#1AATGTAACTGACTTTTTTGAATTC Zcchc11 ACCGGCGTGATAGTGATCTGGATATTCTCGAGAATATCCAG shRNA#2ATCACTATCACGTTTTTTGAATTC Zcchc11 ACCGGGCTTCTGACCTTAATGATGATCTCGAGATCATCATT shRNA#3AAGGTCAGAAGCTTTTTTGAATTC Zcchc11 ACCGGGCAACAGACATGTACAGATAACTCGAGTTATCTGTA shRNA#4CATGTCTGTTGCTTTTTTGAATTC Lin28A ACCGGGAACCCTTCCATGTGCAGCTTTTCGAAGCTGCACAT shRNA GGAAGGGTTCCTTTTTTGAATTCLin28B  CCGGGCCTTGAGTCAATACGGGTAACTCGAGTTACCCGTAT shRNATGACTCAAGGCTTTTTTG

Immunoprecipitation and Western Blotting:

Whole cell lysates were prepared using lysis buffer (20 mM Tris/pH8.0,137 mM NaCl, 1 mM EDTA, 1% Triton X100, 10% Glycerol, 1.5 mM MgCl2, 1 mMDTT, with protease inhibitors (Roche)). Flag-immunoprecipitations weredone using Flag-agarose beads (Sigma) for 90 min at 4° C. Beads werewashed with Buffer containing 300 mM KCl (BC300). Elutions were donewith Flag peptide (Sigma). Anti-Flag-HRP Antibody (Sigma, A8592) wasused at 1:1000 dilution in 5% milk for an hr. Formyc-immunoprecipitation, myc-antibody (Covance, PRB-150C) was added toProtein G-agarose beads (Sigma). Myc-IP was performed overnight at 4° C.Anti-myc antibody was used at 1:1000 in 5% milk. Secondary anti-mouseIgG-HRP (Sigma, A9044) was used in 2.5% milk at 1:10000 dilution for anhr. For endogenous protein western blots, concentration of cell lysateswas measured using Bradford reagent (Biorad).

The following antibodies were used: Lin28A (Cell Signaling, 3978),Lin28B (Cell Signaling, 4196), Zcchc11 (Protein Tech Group, 18980-1-AP),DGCR8 (Protein Tech Group, 10996-1-AP) at 1:1000 and β-Tubulin (AbcamAB6046) at and 1:5000. Secondary anti-rabbit IgG-HRP (Sigma #A9169)secondary antibodies were used at 1:10,000 dilutions in 2.5% milk.Antibody against Zcchc11 (Imgenex, IMX-3587) was used at 1:1,000 and adonkey anti-goat IgG-HRP (Santa Cruz, sc-2033) secondary antibody wasused at 1:5,000. Antibody for Fibrillarin (Abcam, ab18380) was used at1:1,000 in 5% milk, and anti-mouse IgG-HRP (Sigma) was used at 1:10,000in 2.5% milk.

Cell Culture:

HEK293, Hela, H1299, Igrov1, HepG2, T47D, MDA-MB-231, CaCO2, andSK_Mel_28 cells were maintained in DMEM (Gibco, Invitrogen),supplemented with 10% FBS, Pen/Strep, L-Glutamine and Non-essentialAmino Acids (Gibco, Invitrogen). K562 cell line was grown in IMDM(Gibco, Invitrogen) with the same supplements. MCF10A cells containingthe ER-Src fusion protein were grown in DMEM/F12 medium supplementedwith 5% donor HS, 20 ng/ml epidermal growth factor (EGF), 10 mg/mlinsulin, 100 mg/ml hydrocortisone, 1 ng/ml cholera toxin, and 50units/ml pen/strep, with the addition of puromycin. Iliopoulos et al.,2009). To induce transformation, the Src oncogene was activated by theaddition of 1 mM tamoxifen (Sigma, St. Louis) to confluent cellcultures.

Subcellular Fractionation:

Cellular fractionation was done with the NE-PER Nuclear and CytoplasmicExtraction Kit (Pierce). Cells were grown on 3.5 mm dishes and harvestedper manufacturer's instructions. Large-scale fractionation of Nucleoliwas performed as as follows: Cells pellets were resuspended in an equalvolume of buffer A [10 mM Tris, (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 0.5mM DTT, 0.2 mM PMSF]. Cells were incubated for 10 min on the rotator,pelleted and resuspended in 2 vol. of buffer A. Cells were manuallyhomogenized with a pestle, spun down to pellet the nuclei. Nuclei wereresuspended in buffer C [20 mM Tris, (pH 7.9), 25% Glycerol, 0.42M NaCl,1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF] and homogenized witha pestle. Nuclei were incubated on the rotator for 30 min at 4° C., andspun down at 12,000 rpm for 30 min to pellet nucleoli. Nucleoli wereresuspended in buffer E [50 mM Tris, (pH 7.9), 25% Glycerol, 0.5 mMEDTA, 5 mM MgCl₂], homogenized with a pestle and spun down to separatethe soluble and insoluble fractions. The nucleolar pellet fraction wasresuspended in Lysis buffer (20 mM Tris (pH 8.0), 137 mM NaCl, 1 mMEDTA, 1% Triton X100, 10% Glycerol, 1.5 mM MgCl₂, 1 mM DTT, withprotease inhibitors (Roche)].

RNA Extraction and qPCR:

RNA was harvested from cells and from xenograft tumors on day 30 usingTrizol (Invitrogen) per manufacturer's instructions. TaqMan miRNA assays(Applied Biosystems) were used to quantify mature miRNA expression asdescribed previously (Hagan et al., 2009). Pri-miRNA levels wereanalyzed by qPCR. First, gene specific cDNA was made with the reverseprimer for each pri-miRNA, using SuperScriptIII cDNA Synthesis Kit(Invitrogen). cDNA was used for qPCR with iQ SYBRGreen Supermix(BioRad). U6 was used as a normalizer.

Recombinant Lin28A and Lin28B Protein Purification and EMSA:

Transformed BL21-CodonPlus® Competent bacteria (Stratagene) were grownto an OD600 nm of 0.4-0.6. Expression was induced 100 μM IPTG for 2-3hours. Cell pellets were resuspended in cold lysis buffer [20 mMimidazole pH 8.0 in PBS, 0.1% Phenylmethyl sulfonyl fluoride (PMSF)] andsonicated. Cleared lysates were incubated with Ni-NTA beads and after 90min incubation at 4° C. the beads were washed with 80-column-vol. washbuffer [10 mM Tris (pH 7.8), 50 mM imidazole pH 8.0, 500 mM NaCl, 0.1%PMSF). Bound His-tagged proteins were eluted from the column with 1volume elution buffer [10 mM Tris (pH 7.8), 500 mM imidazole pH 8.0, 500mM NaCl, 0.1% fresh PMSF] and dialyzed overnight against BCl00 [20 mMTris-HCl (pH 7.8), 100 mM KCl, 0.2 mM EDTA, 10% glycerol]. EMSA withend-labeled synthetic pre-let-7 RNA was performed as described butwithout competitor yeast tRNA (Piskounova et al., 2008). Briefly,reactions were set up in binding buffer [50 mM Tris, (pH7.5), 100 mMNaCl, 10 mM βMe, 20U RNaseIN (Promega)] with 0.5 nM end-labeledpre-let-7g and incubated for 45 min at room temperature. Bound complexeswere resolved on native 5% polyacrylamide gels and visualized byradiography. Band intensities of scanned gels were quantified usingImageJ software and used to calculate percentage of probe bound.Graph-Pad Prism was used to plot the data. For both recombinant Lin28Aand Lin288B, the percent active protein was EMSA with purifiedHis-Lin28A/B was performed as described but without competitor yeasttRNA. Piskounova et al., 2008. Complexes were resolved on native 3.5% or5% polyacrylamide gels and visualized by autoradiography. Bandintensities of scanned gels were quantified using ImageJ software andused to calculate percentage of probe bound. Graph-Pad Prism was used toplot data. Percent active protein was determined using stoichiometricbinding reactions as described in Ryder et al. (2008). Hills equationfor specific binding with one site, Y=Bmax*X̂h=∂KD̂h+X̂M

, was used to calculate KD.

RNA-Immunoprecipitation (RIP):

Hela cells were transfected with Flag-constructs of Lin28A and Lin28B,or empty Flag-plasmid for 48 hr. Flag-immunoprecipitations were doneusing Flag-agarose beads (Sigma) for 90 min at 4° C. Beads were washedwith BC500. RNA was eluted from the beads with Trizol. RNA was extractedfollowing manufacturer's protocol. Pri-microRNA levels were analyzed byq.RT-PCR. Error was shown as SEM, n=3.

Immunofluorescence:

Cells were grown on coverslips for 24-48 hr, before fixing with 4%paraformaldehyde for 20 min at room temperature. Cells were then blockedand permeabilized with 5% serum with 0.2% Triton for 20 min at roomtemperature. Cells were incubated at 4° C. overnight with primaryantibodies at 1:400 dilutions. The next day, cells were washed with PBSand incubated with the secondary antibodies at 1:400 dilution for 1 hr(Invitrogen, Anti-mouse A21202, Anti-Rabbit A21207), in the dark at roomtemperature. The cover slips were mounted with Vectashield mountingsolution with DAPI (VectorLabs). For GFP-fusions, the Topo CT-GFPcloning kit (Invitrogen) was used. Cells were transfected withGFP-fusion constructs, and after 48 hr fixed as described above. Cellswere washed with PBS three times, the last wash contained 0.2% Triton.Coverslips were mounted as described herein.

Phase-Contrast Images:

In order to study the morphological changes and phenotypictransformation of MCF10A ER-Src TAM-induced (36 hr) cells,phase-contrast pictures were taken in a microscope (10× objective).Furthermore, phase-contrast pictures were taken MCF10A ER-Src cellsinduced by TAM for 36 h and simultaneously treated with an siRNA againstLin28B (siLin28B) (100 nM), a monoclonal antibody against IL6 (Ab-IL6)(2 ug/ml) (Mab206, R&D Systems) or a siRNA against Zcchc11 (siZcchc11#1)(100 nM). Cell morphology was assessed by phase contrast microscopy (10×objective), and percentage of transformed ER-Src cells was calculated byevaluation of cell morphology by Metamorph v5.0 software.

Colony Formation Assay:

MDA-MB-231 cells and T47D cells were transfected with different siRNAsfor 48 hr. Triplicate samples of 105 cells from each cell line weremixed 4:1 (v/v) with 2.0% agarose in growth medium for a finalconcentration of 0.4% agarose. The cell mixture was plated on top of asolidified layer of 0.5% agarose in growth medium. Cells were fed every6 to 7 days with growth medium containing 0.4% agarose. The number ofcolonies was counted after 20 days.

MCF10A ER-Src transformed cells, MDA-MB-231 cells and T47D cells weretransfected with different siRNAs for 48 hr. The siRNAs used in thisexperiment were the following: i) siRNA negative control (siRNA NC) (100nM), (cat no. AM4611, Ambion Inc); ii) siRNA against Zcchc11(siZcchc11#1) (100 nM) (cat no. s23551, Ambion Inc); iii) siRNA againstZcchc11 (siZcchc11#2) (100 nM) (cat no. s23553, Ambion Inc); iv) siRNAagainst Lin28B (siLin28B) (100 nM) (cat no. s52477, Ambion Inc); v)siRNA against Lin28A (siLin28A) (100 nM) (cat no. s36195, Ambion Inc).Then, triplicate samples of 10⁵ cells from each cell line were mixed 4:1(v/v) with 2.0% agarose in growth medium for a final concentration of0.4% agarose. The cell mixture was plated on top of a solidified layerof 0.5% agarose in growth medium. Cells were fed every 6 to 7 days withgrowth medium containing 0.4% agarose. The number of colonies wascounted after 20 days. The experiment was repeated thrice and thestatistical significance was calculated using Student's t test.

Interleukin 6 ELISA Assay:

The concentration of interleukin 6 released to the supernatant of MCF10AER-Src TAM-treated (36 hr) cells treated together with 100 nM siRNA NC,or siZcchc11#1 or siZcchc11#2 or siLin28B was measured via IL6 ELISAassays (cat no. D6050), according to manufacturer instructions (R&DSystems).

Invasion Assays:

invasion assays were performed in MDA-MB-231, and T47D breast cancercells were transfected with different siRNAs for 16 hr. Invasion ofmatrigel was conducted by using standardized conditions with BDBioCoatgrowth factor-reduced MATRIGEL invasion chambers (PharMingen). Assayswere conducted per manufacturer's protocol, using 10% FBS aschemoattractant. Noninvading cells on the top side of the membrane wereremoved, whereas invading cells were fixed and stained with40-6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc.), 16 hrpost-seeding.

Mouse Experiments:

(a) 5×10⁶ MCF10A ER-Src TAM-treated (36 h) cells were injectedsubcutaneously in the right flank of athymic nude mice (Charles RiverLaboratories). Tumor growth was monitored every 5 days and tumor volumeswere calculated by the equation V(mm³)=a×b2/2, where a is the largestdiameter and b is the perpendicular diameter. When the tumors reached asize of ˜100 mm3 (day 15) were randomly distributed in four groups (5mice/group). The first group was used as control (non-treated), thesecond group was i.p treated with 5 mg/kg siRNA negative control, thethird group was i.p treated with 5 mg/kg siZcchc11#1 and the fourthgroup was i.p. treated with 5 mg/kg siLin28B. Tumor volumes weremonitored for 45 days.

(b) 2×10⁶ MDA-MB-231 cells were injected subcutaneously in the rightflank of athymic nude mice (Charles River Laboratories). Tumor growthwas monitored every five days. When the tumors reached a size of ˜100mm3 (day 15) were randomly distributed in five groups (5 mice/group).The first group was used as control (non-treated), the second group wasi.p treated with 5 mg/kg siRNA negative control, the third group was i.ptreated with 5 mg/kg siZcchc11#1 and the fourth group was i.p. treatedwith 5 mg/kg siLin28B and the fifth group was i.p. treated with 5 mg/kglet-7a microRNA. Tumor volumes were monitored for 45 days.

(c) 2×10⁶ T47D cells were injected subcutaneously in the right flank ofathymic nude mice (Charles River Laboratories). Tumor growth wasmonitored every 5 days. When the tumors reached a size of ˜100 mm³ (day15) were randomly distributed in five groups (5 mice/group). The firstgroup was used as control (non-treated), the second group was i.ptreated with 5 mg/kg siRNA negative control, the third group was i.ptreated with 5 mg/kg siZcchc11#1 and the fourth group was i.p. treatedwith 5 mg/kg siLin28A and the fifth group was i.p. treated with 5 mg/kglet-7a microRNA. Tumor volumes were monitored for 45 days.

(d) 10⁶ HepG2 cells were injected subcutaneously in the right flank ofathymic nude mice (Charles River Laboratories). Tumor growth wasmonitored every five days. When the tumors reached a size of ˜200 mm3(day 15) were randomly distributed in four groups (5 mice/group). Thefirst group was used as control (non-treated), the second group was i.ptreated with 5 mg/kg siRNA negative control, the third group was i.ptreated with 5 mg/kg siZcchc11#1 and the fourth group was i.p. treatedwith 5 mg/kg siLin28B. Tumor volumes were monitored for 45 days.

(e) 5×10⁶ H1299 cells were injected subcutaneously in the right flank ofathymic nude mice (Charles River Laboratories). Tumor growth wasmonitored every 5 days. When the tumors reached a size of ˜200 mm3 (day15) were randomly distributed in four groups (5 mice/group). The firstgroup was used as control (non-treated), the second group was i.ptreated with 5 mg/kg siRNA negative control, the third group was i.ptreated with 5 mg/kg siZcchc11#1 and the fourth group was i.p. treatedwith 5 mg/kg siLin28B. Tumor volumes were monitored for 45 days.

(f) 2×10⁶ Igrov1 cells were injected subcutaneously in the right flankof athymic nude mice (Charles River Laboratories). Tumor growth wasmonitored every five days. When the tumors reached a size of ˜180 mm3(day 15) were randomly distributed in four groups (5 mice/group). Thefirst group was used as control (non-treated), the second group was i.ptreated with 5 mg/kg siRNA negative control, the third group was i.ptreated with 5 mg/kg siZcchc11#1 and the fourth group was i.p. treatedwith 5 mg/kg siLin28A. Tumor volumes were monitored for 45 days.

(g) 5×10⁶ SK_MEL_28 melanoma cells were injected subcutaneously in theright flank of athymic nude mice (Charles River Laboratories). Tumorgrowth was monitored every five days. When the tumors reached a size of˜200 mm3 (day 15) were randomly distributed in four groups (5mice/group). The first group was used as control (non-treated), thesecond group was i.p treated with 5 mg/kg siRNA negative control, thethird group was i.p treated with 5 mg/kg siZcchc11#1 and the fourthgroup was i.p. treated with 5 mg/kg siLin28B. Tumor volumes weremonitored for 45 days.

(h) 5×10⁶ CaCO2 colon cancer cells were injected subcutaneously in theright flank of athymic nude mice (Charles River Laboratories). Tumorgrowth was monitored every five days. When the tumors reached a size of˜180 mm3 (day 15) were randomly distributed in four groups (5mice/group). The first group was used as control (non-treated), thesecond group was i.p treated with 5 mg/kg siRNA negative control, thethird group was i.p treated with 5 mg/kg siZcchc11#1 and the fourthgroup was i.p. treated with 5 mg/kg siLin28B. Tumor volumes weremonitored for 45 days.

All the experiments described here used In Vivo Ready siRNAs from AmbionInc which are high-quality siRNAs that are purified especially forintroduction into animals. Each siRNA strand is individually purified byHPLC, desalted, and annealed with its complementary strand. In VivoReady siRNAs are then further purified using a process that removesexcess salt via a semi-permeable membrane. The result is a highly puresiRNA with minimal salt content, suitable for in vivo applications.These siRNAs are then filtered through a 0.2-μm pre-sterilized filterand tested for the presence of endotoxin. Next, we mixed the siRNAs withInvivofectamine 2.0 liposomes (Ambion Inc) and injected them in mice ina volume of 100 ul using a 20 G needle. The experiments described abovewere performed in accordance with Dana-Farber Institutional Animal Careand Use Committee procedures and guidelines.

Real-time RT-PCR Analysis for Human Cancer Tissues:

Real-time RT-PCR was performed to determine the expression levels oflet-7a in human colon and breast normal tissues and carcinomas. RNA wasisolated, using Trizol (15596-026, Invitrogen). Reverse Transcriptionwas carried out using the Universal cDNA synthesis kit (203300).Real-time PCR was carried out in triplicate using the SYBR Green mastermix (203450) and primer for let-7a (204775, Exiqon) in a CFX384 RealTime PCR detection system (Bio-Rad). Let-7a expression levels werenormalized to the levels of U6 snRNA (203907, Exiqon). Furthermore, RNAswere purchased from Origene from the following cancer tissues (8 renalcell carcinomas, 8 hepatocellular carcinomas, 8 squamous cell lungcarcinomas, 8 ovarial adenocarcinomas, 8 prostate adenocarcinomas, 8papillary thyroid carcinomas) and were used to test Lin28A and Lin28BmRNA expression levels. Real time RT-PCR was employed to determine theexpression levels of Lin28A and Lin28B. Reverse Transcription wascarried out using the Retroscript Kit (AM1710, Applied Biosystems). Realtime PCR for was carried out using IQ SYBR Green supermix (170-8882,Bio-Rad). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used asthe internal control.

NF-kB/p65 ActivELISA Assay:

The NF-kB/p65 ActivELISA Kit measured nuclear p65 levels in nuclearprotein lysates derived from 12 breast cancer tissues purchased from AMSBiotechnology Inc and Biochain Inc. The anti-p65 antibody coated platecaptures free p65 and the amount of bound p65 is detected by adding asecond anti-p65 antibody followed by alkaline phosphatase(AKP)-conjugated secondary antibody using colorimetric detection in anELISA plate reader at absorbance 405 nm. Each sample was loaded ontriplicate and data are presented as mean±SD.

In Situ microRNA Hybridization:

Double-DIG labeled Mircury LNA Detection probe for the detection ofhsa-let-7 (1800-15, Exiqon) by in situ hybridization, was used aspreviously described (Iliopoulos et al., 2009) with modifications.Sections of colon adenocarcinomas and adjacent uninvolved tissues weredeparaffinized with xylene (3×5 min), followed by treatment with serialdilutions of ethanol (3×100%, 2×96% and 3×70%) and by two changes ofDEPC-PBS. Tissues were then digested with proteinase K (15 μg/ml) for 20min at 37° C., rinsed with 3×DEPC-PBS. Sections were dehydrated with2×70%, 2×96% and 2×100% ethanol, air-dried and hybridized for 1 hourwith the hsa-let-7 probe (40 nM) or the double-DIG labeled U6 ControlProbe (99002-15) diluted in microRNA ISH buffer (90000, Exiqon), at 54°C. Following hybridization, sections were rinsed twice with 5×SSC,2×1×SSC and 3×0.2×SSC, 5 min each, at 54° C., and PBS. The slides wereincubated with blocking solution (11585762001, Roche) for 15 min andthen with anti-DIG antibody (1:800) in 2% sheep serum (013-000-121,Jackson Immunoresearch) blocking solution for 1 hour, at RT. Followingthree washes with PBS-T (PBS, 0.1% Tween-20), slides were incubated withthe AP substrate buffer (NBT-BCIP tablet [11697471001, Roche] in 10 ml0.2 mM Levamisole [31742, Fluka]) for 2 hr at 30° C. in the dark. Thereaction was stopped with two washes of AP stop solution (50 mMTris-HCl, 150 mM NaCl, 10 mM KCl) and 2 washes with water. Tissues werecounter stained with Nuclear Fast Red for 1 min and rinsed with water.At the end, sections were dehydrated with 2×70%, 2×96% and 2×100%ethanol and mounted with coverslips in Eukitt mounting medium (361894G,VWR). Images were captured with a Nikon 80i Upright Microscope equippedwith a Nikon Digital Sight DS-Fi1 color camera, using the NIS-Elementsimage acquisition software. All images were captured and processed usingidentical settings.

Immunohistochemistry:

Sections of the colon adenocarcinomas and adjacent uninvolved tissueswere deparaffinised with xylene (3×5 min) followed by treatment withserial dilutions of ethanol (100%, 100%, 95% and 95%, 10 min each) andby two changes of ddH2O. Antigen unmasking was achieved by boiling theslides (95-99° C.) for 10 min, in 10 mM sodium citrate, pH 6.0. Sectionswere then rinsed three times with ddH2O, immersed in 3% H₂O₂ for 10 min,washed twice with ddH2O and once with TBS-T (TBS, 0.1% Tween-20) andblocked for 1 hour with blocking solution (5% normal goat serum [5425,Cell Signaling Technology] in TBS-T). Lin28A (3978, Cell SignalingTechnology) and Lin28B (LS-B3423, LSBio) antibodies were diluted 1:50 insignal stain antibody diluent (8112, Cell Signaling Technology) and1:100 in blocking solution respectively, and incubated with the sectionsovernight at 4° C. Following incubation with the antibodies, sectionswere washed three times, 5 min each, with TBS-T and incubated for 1 hourat room temperature with anti-rabbit biotin antibody (LS-D1, LSBio)diluted in blocking solution (1:300). Sections were washed three times,5 min each, with TBS-T, incubated with the Vectastain ABC-AP reagent(AK-5000) for 30 min, washed and stained with the Vector Red AlkalinePhosphatase Substrate Kit (SK-5100), and with the hematoxylin QScounterstain (H-3404, Vector Laboratories). Finally tissues weredehydrated and mounted in Eukitt medium.

Human Tissues and RNAs:

Thirty normal colon tissues and 45 colon adenocarcinomas were collectedfrom the translational pathology core laboratory of the Department ofPathology at UCLA. All subjects gave informed consent, and the study wasapproved by the UCLA Institutional Review Board. RNAs from twelve normalmammary tissues and 33 breast cancer tissues were purchased from OrigeneInc. The ER, PR, and HER2 status for each of these breast carcinomas wasknown. Additional RNAs were purchased from Origene (8 each of renal cellcarcinomas, hepatocellular carcinomas, squamous cell lung carcinomas,ovarial adenocarcinomas, prostate adenocarcinomas, papillary thyroidcarcinomas).

Example 2. Molecular Characterization of Lin28/TUTase/Let-7 Interactions

Cloning:

All mZcchc11 mammalian-expression mutants were cloned into the XhoI andSalI sites of pBK_2× Flag EF1 vector. C326/329A mutant Zcchc11 wasgenerated by site-directed mutagenesis using the QuickChange kit(Stratagene). hZcchc6 was amplified from HEK293 cDNA and cloned into theHindIII and BamHI sites of pFLAG-CMV2 (Sigma). For recombinant proteinexpression ΔPneumoG/C mZcchc11 was cloned into the SalI and NotI sitesof pETDUET-1. Recombinant mZcchc11 C2H2 was cloned into the EcoRI andHindIII sites of pETDUET-1. Expression constructs for Flag-m.Lin28A,Flag-Lin28A, Flag-Lin28B, recombinant His-Lin28A, Flag-hZcchc11wild-type and D1026/1028A mutant were described previously(Hagan et al.,2009; Piskounova et al., 2011; Piskounova et al., 2008; Viswanathan etal., 2008). Cloning primers are listed in Supplementary Table 3:

TABLE 3 TUTase Lin28 cloning primers mZcchc11 TGCCGCCTCGAGGTTTCTATGGATAAAAGGAAGAGTGAA ΔPneumo G XhoI F mZcchc11  TGCCGCCTCGAGCTTCGGTCTCTTCCATCTCCTT N-PAP (ΔC2H2)  XhoI F mZcchc11  TGCCGCCTCGAGTGCTTACTTGGAAGTTGGATTGAAGG ΔTftF4 SalI F mZcchc11  TGCCGCGTCGACTTGCTTTAAGTCACTGCCTCCAC N-PAP SalI ft mZcchc11  TGCCGCCTCGAGTCCCAGGAATTATATTATGTGTTTGAT PUP XhoI  AAGTT F mZcchc11  TGCCGCGTCGACTTCTGAAGACTGTCTGGTCCTTATG PUP (ΔC) SalI ft mZcchc11  GTCGACGTCACGGGAGTCTTTTTCTTCTTC ΔCCHC3 SalI ft mZcchc11  TGCCGCGTCGACATGATTCAAGTCAAAAGGATCTTCAAT ΔCCHC2 TG SalI ft mZcchc11 GTCGACTCTCTGCGGAATCTGCTTCCC ΔPAP- Assoc  SalI ft mZcchc11 TGCCGCTATCTAGCCAAACTTGCCTTAATTCACATT C326/329A  F mZcchc11 AATGTGAATTAAGGCAAGTTTGGCTAGATAGCGGCA C326/329A  ft mZcchc11 CGATGCGTCGACGACTACAAGGATGACGATGACAAAGTT ΔPneumo  TCTA G/C SalI Flag TGGATAAAAGGAAGAGTGAA recomb. F mZcchc11 GCGGCCGCTTCTGAAGACTGTCTGGTCCTTATG ΔPneumo  G/C NotI Flag recomb.  ftmZcchc11  GTAGCGGAATTCGTTTGTTTCTATGGATAAAAGGAAGAG r.C2H2  TGAA EcoftI FmZcchc11  GTCGATAAGCTTTCCTTGTTCTTTTGCTAACTCAACAAC r.C2H2  HindIII  fthZcchc6  GGTACTGGGCAAAGCTTTGCAG HindIII F hZcchc6 CGTAGCGGATCCTTATGATTCCTGCTGGGTCCTC BamHI ft mZcchc11 GAAGACAGAAACAGACAACCAAG qPCft F mZcchc11  CCTCCAAAGCAAAATCCAGTG qPCft ftmLin28  AGGCGGTGGAGTTCACCTTTAAGA qPCft F mLin28 AGCTTGCATTCCTTGGCATGATGG qPCft ft mActin  CAGAAGGAGATTACTGCTCTGGCTqPCft F mActin  TACTCCTGCTTGCTGATCCACATC qPCft ft mZcchc6 used taqmanprimer set (ABI, Mm00463475_m1)

Immunoprecipitation and Recombinant Protein Production:

Expression plasmids for Flag-Zcchc11, Flag-Zccch6, Flag-Lin28A, orFlag-Lin28B were transfected into HEK293T cells using Lipofectamine 2000(Invitrogen). Cells were harvested in lysis buffer (20 mM Tris-HCl, pH8.0, 137 mM NaCl, 1 mM EDTA, 1% Triton x100, 10% Glycerol, 1.5 mM MgCl2,5 mM DTT, 0.2 mM PMSF). Protein was purified using anti-Flag M2 beads(Sigma), eluted using Flag peptide (Sigma) and confirmed by Western Blotanalysis, with a mouse anti-Flag antibody (Sigma). For recombinantprotein production: Transformed BL21-CodonPlus® Competent bacteria(Stratagene) were grown to an OD600 nm of 0.4-0.6. Recombinant proteinexpression (r.Lin28A, r.Zcchc11, and r.C2H2) was induced with 10004 IPTGfor 2-3 hr. Cell pellets were resuspended in cold lysis buffer [20 mMimidazole pH 8.0 in PBS, 0.1% Phenylmethyl sulfonyl fluoride (PMSF)] andsonicated. Cleared lysates were incubated with Ni-NTA beads and after 90min incubation at 4° C. the beads were washed with 80 bead volumes washbuffer [10 mM Tris (pH 7.8), 50 mM imidazole pH 8.0, 500 mM NaCl, 0.1%PMSF, 1 mM DTT). Bound His-tagged proteins were eluted from the columnwith 1 volume elution buffer [10 mM Tris (pH 7.8), 500 mM imidazole pH8.0, 500 mM NaCl, 1 mM DTT, 0.1% fresh PMSF] and dialyzed overnightagainst BCl00 [20 mM Tris-HCl (pH 7.8), 100 mM KCl, 0.2 mM EDTA, 10%glycerol]. Proteins were further purified by size exclusionchromatography using a Superose 6 gel filtration column (20 mM Tris-HCl(pH 7.8), 500 mM KCl, 0.2 mM EDTA, 0.2% NP40, 10% glycerol) and peakfractions were dialyzed overnight against BCl00 and stored at 4° C.

In Vitro Uridylation Assay:

Purified proteins were incubated with 4 pmol of unlabelled synthetic RNA(Dharmacon) for 1 hour at 37° C. in a 30 μl reaction mixture containing100 mM KCl, 20 mM Tris-HCl pH 7.6, 10% Glycerol, 125 nM [a-³²P]UTP, 3.2mM MgCl2, 40U RNasin ribonuclease inhibitor (Promega). Products wereresolved on 15% denaturing polyacrylamide gels and bands were detectedby autoradiography.

Electrophoretic Mobility Shift Analysis:

EMSA with purified His-Lin28A and His-C2H2 domain of Zcchc11 wasperformed with end-labeled synthetic pre-let-7 as described but withoutcompetitor yeast tRNA (Piskounova et al., 2008). Briefly, reactions wereset up in binding buffer [50 mM Tris, (pH7.5), 100 mM NaCl, 10 mM βMe,20U RNasin (Promega)] with 0.5 nM or 5 nM end-labeled pre-let-7g andincubated for 60 min at room temperature. Bound complexes were resolvedon native 5% polyacrylamide gels and visualized by autoradiography.

In Vivo Knockdowns and Quantitative RT-PCR:

The indicated siRNAs (see Table 4) were reverse transfected in eitherP19 or feeder-free V6.5 mouse embryonic stem cells usingLipofectamine2000 in 6 well plates, according to the manufacturer'sprotocol (Invitrogen).

TABLE 4 siRNAs Lin28 si #1 GGGUUGUGAUGACAGGCAAUU Zcchc11 Si #2GGGCUAAGCUGUGCUAUAU Zcchc11 Si #4 CCAAAGUGCCUAUUGUAAA Zcchc6 Si #2AGAUCAGGCUUCAACGUAA Zcchc6 Si #3 GAAAGUGAGGCGACGGAGA

Total RNA was isolated 60 hours post-transfection using TriZol reagent(Invitrogen). To analyze relative mRNA levels, 2 μg of total RNA wasreverse transcribed using random hexamers and SuperScriptIII(Invitrogen). MiRNAs were reverse transcribed from 10 ng total RNA usinggene-specific stem-loop RT primers (Applied Biosystems). Relative levelsof miRNAs were determined by TaqMan based real-time PCR, snoRNA-142 fornormalization. For quantitative analysis of mRNA levels real-time RT-PCRwas performed with either SYBR green or Taqman assays. Actin was used ascontrol. For global microRNA profiling, the TaqMan Rodent MicroRNA AArray v2.0 was used with 350 ng total RNA as starting material for themultiplex RT with pre-amplification, according to manufacturer'sdirections (Applied Biosystems). The resulting data was normalized tothe U6 snRNA.

1. A composition for inhibiting Lin28A-mediated let-7 repressioncomprising an agent that inhibits the interaction of Lin28A with 3′terminal uridylyl transferase (TUTase) or a functional portion thereof.2. The composition of claim 1, wherein the TUTase is Zcchc11 or Zcchc6.3. A method of inhibiting Lin28A-mediated let-7 repression in a cellcomprising contacting said cell with an inhibitor of TUTase.
 4. Themethod of claim 3, wherein the cell is in a tissue in a subject, andwherein the amount of let-7 in the tissue is increased in the presenceof the inhibitor relative to in the absence of the inhibitor. 5.-7.(canceled)
 8. A method of inhibiting tumor growth or preventing tumor orcancer metastasis in a subject in need thereof comprising administeringa therapeutically effective amount of an inhibitor of a TUTase and apharmaceutically acceptable carrier, whereby tumor growth is inhibitedin the presence of the inhibitor.
 9. (canceled)
 10. The method of claim8, wherein the tumor or cancer in the subject expresses Lin28A.
 11. Themethod of claim 8, further comprising measuring the level of expressionof Lin28A in a in a sample of cancerous tissue obtained from the subjectprior to administering the TUTase inhibitor.
 12. The method of claim 8,wherein the TUTase is Zcchc11 or Zcchc6.
 13. The method of claim 8,wherein the inhibitor inhibits the expression of Zcchc11 or Zcchc6. 14.The method of claim 8, wherein the inhibitor inhibits the activity ofZcchc11 or Zcchc6.
 15. The method of claim 8, wherein the inhibitor isselected from the group consisting of an antibody, a RNA interferencemolecule, a small molecule, a peptide and an aptamer.
 16. The method ofclaim 15, wherein the inhibitor is a RNA interference molecule.
 17. Themethod of claim 16, wherein the RNA interference molecule is ashort-hairpin RNA.
 18. The method of claim 17, wherein the RNAinterference molecule is a short-hairpin RNA directed specificallyagainst the Zcchc11 gene or the Zcchc6 gene.
 19. The method of claim 8,wherein the cancer is colon, breast, ovarian, or prostate cancer.20.-33. (canceled)
 34. The method of claim 8, wherein administering theinhibitor of the TUTase increases the expression level of let-7a levelin the subject tissue is increased by at least 2-fold.
 35. The method ofclaim 8, wherein administering the inhibitor of the TUTase increases theexpression level of let-7a level in the subject by about 2- to about10-fold.